Lawn... ufl..1&u..0.400.r-. -d.41.-.i..3.1..h .....ru I. I .. 0r. .....4. .y . a .. .. 4 L f ...—4r ..,. 48. .. I ... .... t r>.z.4_ ......i. . . .I.... ..44. . .4 _ raw”. 0. .30....) u . .5 . , , . not»? . ....4 0... 0.3.4 ...0. ... .44.? .4 . .4 L: I ..I .I I. Wm3...“..l. p... ....-..Wo 1..-0.31.1.0.0 .. a. ...4 . ... .. .. ... 0.. 0.4...4-4.‘ 91.1..) J. . ’95 Lou: ..IAII- .. .4 .«Iv. ..FF. .r .. . aflfi? - .40“.- .1...I 1. 3.2:.01015. - 334 14 4 0 4.. (M4... , ...... r0..,.I. .... . I. .0... v. 3... .. 144.......3.s .. 00.. -.-n.4....1..0.4..+.1. - .... . 0,- I 003%,”); .0... CM. .‘ * 04“ 0.30 0. 4. . 4 0.". .. ...“ Q4 4 .I.-...00. 0. 01.84.10 -. .0443”. 00.00 > .4.. .I.. «II... 10.. ..0. $0.. o. .0. . . , h ..0. .. 4.» . 4 C. )0. 5.20.4.1... III... 1.4.7... 1:... ..0 , .. ,. #31 I.» ."._-3‘-1.0 .. . 4.4 . 9-00.40? ’0. ' 440", 1.1 ...0 0 O... -44 4. .4111. 0.. 0 Que-..0. I . .I 0. ,.I. , .’ >.. - 0... 0 1.0.4.7.... .0 4 . . .0 0 .u‘ 0’0. I 4n0 0* .m.4 u.» .gd...lt.. ...‘C...«44 4.3.30 4...... I4... I . 1...-0?» .13 11.10. .. 41. .4.. ..Ii-4 .4. «”3... . ..0...- .... I . .- 4. ....4 . .I.-3.0.444 .010- .4. I u .0 (“00.04. . u 0 .0- ltnfivn.‘ 03 I C 100- 0 00:9... 4. ‘u- . 0&1 J. 0.."- J. 0 20>...‘Iu14o4n0 4/09... ...“ .0.I.>o. ... ..‘00- . I .0 0 4,004 .~... 4 ... . .. .l .’-;I ... I. .30. 041.. I .. ...0,“ 4I "4.0.9.0. .... .4 4- 01.34? 5‘.a- . :04 4 . .I A... .0: . ..k. .... .... .2 . .1. ...-l .... ... . .I. .0 . o 9 .0 .11.... ......) 1....4....1.L . ...4’. .... .. -..\. .I- 4 ‘4 .0304. 03.01.00. . ..0 2 any}: ... Tun .I.. . . I .. 1‘. . 4. o . ...-I5}: ”...-.0 . .0. .. fi..o-Vlo. . .0. 4. ...- ; ..4 ... . 3V... ... 0- .4. ... . .,«I.0......I. ..I.«—..0-1.4«0 ..II ..0. .3 00 404.1.- .44.. ..0 040004 ..0 I... .I.-00.0 . o. «1.100,. J4. 0041.01.20.00 0'0 0- J I. .1 0V. .... .4. a. 0. 4. ..‘v .I. 0.. ..0... . -I1.\«.4 .01.. . . ... I ..J, A -1..I_. l . .I ..,.O - ...-«.1031-1. ‘44. .I. .0. 4.0230. n 1410!... 10 .II .. I... o. . . . - .r ...; . . .I.-:40 0004.34.01}! 0.4 ..4 .1: v .. . 0-14.. 0?. $34 I. s ..0. 01. .. ... 3 .4I. 44 a. .00”... ..4‘.‘.. .. .190 ...00. 4.91.»; . 3.4- . 44- .. .114 1.0.. I! ...: .0. t o.- 3.1.1.0. I ...4. ., 0- 4 44.0... - K 1.94%.... .1 4 . 14 s. 1.1. «41,. u ._ ..0“: 4. ......u‘ . .00” .17...- 40.... ...... flurrfiw. «I. 0.4),»... ,.0 . I 1"“ 4.. 00. 0. 0-0 0. 4 . ‘0 I041 . 3 . 0 .‘gt 04 4:00.,1- 001-0400 I s’ u I. 0 ‘1- 0.13002100.“ .4 .4 . 4.1.1 -. .4.. .0 . 0&4 . :10 I 0- 0.10"“ 3 14¢ Q, 1 UMP" «pad! ...-44 . ..1. 2.300340 40.9.0014. . 1L . ,3.) 4... [042.40.10 .. 01‘. +4.. 0 4:. 4 u ..n .0 .. .. .... .LII II .4 ._ ... . .0 .... .- 4..n.A\1...I.4..04..-. 0. I 4 0.. .. ”hr-«w. $41.. . , 3-310“. 0. 43.7101? .5 I. .4 ”001013....“ on. 4, .u ..0-0 3-“..4L., {400.433.} ... Iortl.‘ I116! 50.. .- .I.-.I.. . 4140.31.18..- 3. . .... I 0.10. ... - ..1 .., .043: ... ..r ..000‘. I 100.3. ... 00 .‘0 .42.... o .0000. 40... ..0. . 00410. 0 0 0.3.0 0. 4... 4.40. ..- L .0 .0 .00 .4.. 4I0_ o I 00. 00. f I, ...-Io‘f I. . o. 00 4.0.1.0.. 409.2034,” .I.,44‘ ... .441 . 0000 fins 0. I ... ‘04 .00 00 [‘0 0 40104.; .10 I. ”I0 . I .1-“0‘. 1.01. 4. .. 4. ob|_, .0'40‘04400 . 0a. .0 J‘n- . I. 0- .40 . . ..Il-p..0. ...: c. .0. 004-04 ..44ahuu:ur.d. .. x. ...0: PPM ”4413?? 3.1.0..) ... . .- 4. . J. .....ua‘h‘o «4.0..JOK? ..J. 6 .14. . .... . 2 . ...-4 ...: .. ...0. ...NIU... .. ..I. ... =- .0171!“ . .0. . .17. «H -- 4...... 4 . ..- .g.‘0¢. ...c: 4.0:... ..0... “.4.? I531 1 ...- 3.84.1... 4 41 -..-2.91.5.1... . :1. 40 1...-24... .4413. . . :2... ..N....... . I4, ... I. . .1 .o I. . .. ..I 4 .1... . I. 4 .12.... I. 1. 4.. 4.4;“.10..u,..4. ‘44. .....04 .63914010-01 (...... 44-1. .I."? 0-0 . ...... «1....40004 ...-40. ,4. 4 . .1000”; 0 .0. .. ...Q‘. . I 4 ...0 0 .1... 10.. 4... . .4.-4 é..- ...0-«44...... l 4.. ..fit.. ...-R ...M ..U. .4. 4.... - .. ":4.- .1. .....i- .401... ...me ... ...-.... .... .. .. .... L .I....I n.1- 10H... ..- .. -... to 0 040.44 40‘00 .44 0.44 .04 '00. .4 5| R0 0’4. .00.. ..I-OZ.‘¢J ,.0 I- 000013'4 ... 0.400. 10.0. r. . .444!- I ,.- 0. 040 0‘41 '70.}...‘0. ‘ 5.4.0,.00u410000. I. ..o‘. L? 0]; 10 31.9.2.0 0 0. a ..10- .1 14.1%.32..h1.=..4 4 1. I ..I .Ir..| ‘3.- ... .0 .0. 0 ... .1. ...0. 24.0.1.0}... 0.00.. .. ...0...4¢..... 0.4. 340-. . .I.- ..r .0... . .- a. . | 1..... 4 $4.000. 0. .44 ,I-II' 0.40 4-04.... .4 4.01.? 9%.}... 00. ......A: o. .40. 4.. I4 .00. .930 :01.- ..o! ..,-4 «4 1.. 4. ..4: S. . ._ 04 £0.00. ... .0407... I 4 ..U... .0. . o. .0 0. n .4.. .4 4.04.. I: 0. 4.44.91.04.32? V. on“... .4. 1.1., . .0 ... 90.1.. ...0 .4. 430““. 7.5-0.1... . ... .0”... . -...¥4..- i... I100 4.0 41.40 .03. .00.!. ...0-44 4 I4- .. . ., . fl. . . fist-14.0 fl . .. .913“. h 4 I. .00 .I. . 0L..wo. 0 0 I014, 4 .0 0‘ .mn 0.100. ,.0 . ...-01., ...0. ,4 ..>. 4’. ‘4».«49. I... .4. 4. ..0... . . I. .9001.- A... «II . €0.04 ....4.- 0 .4 .. . 1 0 c ...0 11.1.1.4 0‘ . I.) 001‘ 0. 01.14"”...3400: .41. 9 14-40 1 09-41:! 4 ..‘4....4. ..0-..0 . II 9. .404 ..0. . .4 4. I ..l.l- . .. :40... ..0-4.0.. 3304!: 4. ..0. . I I. .... . I... ...8..1u..u4«?0- . . .4112... “43W"..4010.1..444......-.u0r: . ... .I.-1.140.. 3.44:3”!!! 304.1 $4040.43. 4.1 . .- .3741111. .31.“ .. its... ...0 31...... I. . I... . ........ ... .. . . J 14......4? .I.-a. : .. . .. ......4 ..0). .4 .. : 34.4 . $4.10 :0» 0.0 .0." 0 00.10.04 0‘43... .0210 I... ...-“:0 I, 1 «‘4 6...: 3003......1 . 3 ..4 0-. 4 4. II, 40 . o 4. 3001'. I, 0 4.4.1.0 I. . . ..0‘ 0. .07 0 7,31 . 4 I .u ..,. . >0. ...0- ,II I b: I... . 4. 0|. ..a. (b .10.... £440 .1 mar-.5. 04. . ...: ...-L. ,. - 4.0 . .4.-U10... .. 31.......1-3».4..141.m1..«-3.11.51.2.10... o - 0.1 I... . ,4! ,,.. ...-,- 1.903.001.414... .. - 1 . .. 2.1 1...}.0’! Hafiz“... :11. .”,44... - .4. .. .4... ...-.54 I .. . . .. ...0-...... I. 1.... .. to... 0-). .I. .. 4-4. . “11.1.1... . t . . . .. . . . 431.21.330 14.34. .3211 '44- 0. 00.?31' m'ao 4. Ixmo. 00””.«3 .I.! 4.00- .0. .. 0-I0. .0 .. o 004 ...: .‘..- 10.0."..qu ,.’100n0¢-...04 330-.- . .... .. 4 .I.-.4. \O r. ... 4. 0409.001 ...-4.40.1.5... .« no . . , -1.I...0..4.I..I.4., 04%.: .I.... .v4.". .‘4‘ \b'!‘. 12,400 102:; ... . 400-. ..‘.,LOI‘o0. ....0 3%.. . .100; o 40 0 .4 . I 0 0.400.410 . 09. I 1..-s! . .0 o .4 .0 .4 .4 I I. 0.0 . .... .40 44 .II.L,0\.04‘4.0 0‘. #2894741. “K101221i ... 4.. 3.0.14: . 4.01:..- . 4.-.... 1..... 4... -4. . .... . I . \- 80..s: .. I 41.3.12... .P... I... ..0-JR! a I . .... 1..... .o ....14 .44 I .I- . a. 1:4 .4 I. 0.. a“; .. .I. ...-x. .. 0.1... .. 210.. 0.. ..0-0.100. ,- t? .. .0. 4 0.2- n . 904. 4.4.5.91... .3’. .000... .. 1-0-3011‘. .04 I.- - 00- 100. .4. .4. 134403.... . .540... . . .. . . . . ...! ..40I.1.II . >. 0 ......«04 04 .Jgnfé. 5. ’124.‘33.,-t.3941...«.-2L01 «..0-14 I... . ... 0‘1. .4 0. .4. .I....- 1 (240.... 1.41.4... . .. 4 .4 o. 0-1 :3. p .I. ..0. . o 00. .000 . 40 I040. . ... ...0. \R>.l.... . _ . .. . 4 1,... 30.4.00... ...-"o. 10.. :4. 0 44?... '3: o 1 .0 .0 4. 4 .- ..,. I ‘00.‘..¢F3..- ...—4. .00 ...... ...0. I ... 30.2,. 10.0 4 ..0-0.5.. . .11.... 0., ..10¢.I.II . .. .0 044...... ) 910’. o...- ... motif. ...0, . . ... .09..)4-2: 44.4.. o. I 4. {.04. . 40W“; {43 l‘. 400.. I.‘1.I.00.Ao.!04.‘0.42. 4 4. «05140-0 444440.000! 404 ...0 01.4.10 3...: I “..0. I. 00.4 040th"; 10.0. 0. . .0300. v.1 440- . ... .0! 4. .o 00.. 1.2.1.04...’ . .. , 0.004 .405 ..l 4.. .10 ..i 0.0.2.. .0140 1.4 . a. 45. 1...-.0... flog-4 .4- ... .30 «.4 ...0, 01......4-‘1440-24- .- .144 n 44 ..0 .. .4 t .III.0I-..-14. .I.. . . ... -. x. I... .0 ...-.0 420.3. 31.1.4... .4 810-. a.» . , ..0: 145...»; .I.-...I- .4 gm“... 0.1010 .0? ...0. ...»“azd ..lo 4 P. 00-3.. 0.! 204 4 02.521.47.04 .40 . 4 41- ,14 ...0 .4 ..,-4.... ... too. 344... ,.. )0... ..,:0 .. .. I 00.7.41,- .0. . I41 4. > s. 4 . -. , ...4 I .. 5-4-. .. .. .04. 4.3.4.0....00304143 . 41.44.... .!00.11’ a. {[4133 1.4 ...04«.§VI e113: ...idw.~4;44|410§.1${1«.80«.5 41....1: ...-.I.}...cs- .... 0.. “4.1. .32}? .4 44:40.31 ...-10...“! .11."..«81 .04. ... “.1501... , 1.3.1I3..1..3.10.I- 34.4.0 .0303. 0...... ...-.0". $.0-14lov 1.4.1 . £1.00... .I. 4 01.... «(W4 ...... ...0. .L. 00 1..-4.1... ..0- 1.. 30-00044!- ..- 59...“. .0 4 :4... 4. . .1 8-- 4.3.4.... . ..)I r. «1..-......000. Ln... . .u _ $000. ... .04 a... . L01.’ .0000 $20. 0404440.. 0 Pl {0-50- 1 4100.“ o. 3501.. .0. 1. w 0.44’1d’105’00444m-u .13.. 0.3.. I. 0.. I... . 40-. . . . . . 0 ..0. .144... .0419}. 0.0.0.000 3.2. $4 . Ivy. -..I.. 04g". .-..v..d...4.. 4100.41.44.3- .1 0.... ... It .0. 04 o .I. . I? ah..- ... ,Jm-0..-.. .o 0.. «41.3.4... .. .0.,. I.\. 4.0.. ...I. . . . . .. . . III... . . . .. .I.; ... .I .113 24....) 1 f...‘ 2.00.4... 3418...}. 4-00 ...-fl 1. Jaw-100.453.4411. 040. 4000-34. 44..- 090. 0.. 0.. 1... 4. 1m»... .I.-00 ..0... 0.11.4?! I... . .. .. . . ...-...0. ..0. . . .0 4. I... . .. 0. . L .1. ...14 24. .4 . .1322?" .I........tn-00~0.0 .31 4 .02.... 8.2. ... It... .. .03. 4.1. ... 4. .4.. . - ...0. - .01 400“.” .1. ,.0. .4... -4.. 30.0 .0... . T. . :02... . ...0-4 ‘1.»I.. 9. . I .. , ,. ..4 4 000M. .0 ....- I .00 .4 44“. 0. 110.041.0000. 04 I 0. 3:44.: 0.... II... 1:01.. . 1.. 3 I . I .‘ .01 3"... 40000 . 0* 00 0.. u. . ..I4 .III- . .400 00.040 0. $0 I 00...: 00.440.0001 o 01.1 0.. 0 I. I 01100 4. 0.1-. ..000. .001. I. ,4 - I . 020.1: 3... . 00 ..I 6.4 .- . o.- 4 D 01100’I‘000 ‘ 0;. 7:03.05, ..1A.IW.-00"..W. «4.4.1001 -.«, 040 L 0.0.. .I. t ‘0. .. ..h .13. I420 .- .I.-04 . . .v a If... .- 0n 10 ...0 . 4.. «1.... 41.00. .0 .I. 4.01.0310 l.. a.“ .I.-o .I.! 000.0 4-‘ .512221141‘014 .04“... ..0) 40 VMII 20000,... 3.0- . ‘2 000 00.0.0 .4.. «a: IJ."§.J..3-‘014.45300 «(... 20.0«0. ..2... .4, l. ..w0 c1404 .- 0‘00 401' 00-53-1410 ..0-.31.? 4.4 ..4I...44.M4,.. . 0.0- - .1 0 a 3. . ... I. .0!) \XII .4 .. .0. «‘0 ,. .51....ru0..oo‘nfl...v-I aw. 0-22.01 .fl-‘H’TE .0 0,-0.0 1.9.0400. 00 323.40. 4 .20194‘2- .3. 4. .n 0. «I10 004... 1..., 0-0. 0-. .00.. ’0 I ..0. :10. 40'. 0|. 0 01009-13... .. 0.. .I 4,0 00-1H10 00.0. (..0; . .I.. .10 .4...” 0 .04“. .1 .4011} r V 9.0004 ‘00-‘45. ’4" $10...) $4. 4.0.4341... hcunovin 4... I»: ... can. 904...“. 4 I44... ’44 “1.000 0.: 03.9.0 '0 . ... .. ..u .0 ‘0.I I ... Iiw 4 I . 01:1.» 0249:. . 4. 44. . .. \.-0. .4. 40". 0 . 4 . ... o. I . {1.. . . . . . .I.... .0 4 a... .4 ... ... u. 1).:I ). ....“ 1.40"”. l-W.L,........ 24.13. 1.110 «3.5.. 20114.“? . ..0 .. 33.1.1.4”‘4309. 4 I .. .L. ...-0 ...I . 0.. . 1!... 04...; 4.. .2. 1., .. . 2.9.5131... . . . . .. 0.. ..0 . 04. 391.404.2313 1 } .1fl-t-13 413911.10. 03 O: I 00.- qudbiI. ..0. v ...0 -I0 .L.-\0. 5...... 3-40-0112. I .Onl-IV... .74. .-II‘, .44 02...!" III 001.4000 .... o. . . . .. \4.‘ 0.. -. 441‘1.I. .d.\1- .0 .. ..0... 1.0. 44 44.0, «40000 041 00.40”“ J Wu“. .. 4... . -..-Nu . .0“ 3:1 1.1.10.9 «.... 4.. Thu-m4..." ..0. ..0... I .... 4.... .- cs... .0 04... 0- I0. .144 ..0-...0 . .400..- . .0 ,. . 0 .4 . ,9. . ... «3!. 0.1... 0...- . .VI......--. 4... a1... 1.0.4 u. 04. .4.4...v1\0 0! ...)07 ..,-.0 40“}: w. . 130.... .310. 40-. r .u.44.. I. 4.. 4 .II 14.1.... .., 1...... .- .:3 I. ... .I.. “2.1. - I4. , . . . . ..0 0. . .00. 01...». I. .... .1 0.. 4!*....o..... 40107 .330“). £49.” .“w. cl 4.. 00.. a... can. 4.04.004 . I 41...1.-.!.:..,I.4 n .. 1.0.51.2. ' 0.44. 0.. I004... 1.3.1.4.!9: 1.. . 342.302, . . . . .3400" ..0. .1411; I 4 ... ...-....r 4.041114. .0114. 1.420.010? ...0-01.44.01.410. . . ..4 ., .9} “no. ..0? . dim-‘0 01... .11- .042 000.0. - ......Q. .1 4. '0 IL, I .0 .7 0017.31. :1 tip... ,0. £3.00 ..4 40-..ng 4 44.4.0. 4 0-. 41,0. - .00 .. ., , . . .00..- 0 . ... ... .I. , ...-VII .. 4. .ILQI .01 190,530. ...-41.1 . . 30030.1 "H.442 404-3301....41 . ...-4.“.0 .0... .50.». .V! 40... 4.1 4. 4:. ...-.3...- 419.1440...-01 >4! .1041 .I. .040. 390.141. \.....- ......0 2.31.3: .0 2.. .1 4.31.0.0 I. . .319. .I.... 04 04034.0. [4414..- ..-.—1 ...0’3-410.4b..r?..\.400.,0. . 1...-04100:“ 1...« 4351.143.“ 91.21.1401 8- 304.440“; .. .4 0.0.04 .140... - 0. 0.. 0 .4... . ...-... .01! ...-...0... .I. . . . .... 30.... ..33 .4. .3kw44.....a4.... . . .. . .5. . ...-... 12...... .o ....V...01.. _ ...-s... . . ... 0.... . 410 I039 .04...“ o. ....Jbon.1\‘vl..1‘0o0.1...... “..0-... ...0 ...-.11....3. .. 1.0.0.4. 0:14.23”? .fla 4-4.1 o. .4.. ,.0? I 0- 2.... 1..?0’0- ...-..0- .. .4. . I... . .0. . . .. . . . .04 .44. . o0fi ...-:1. .I..- ...444. .I .4.-... 2g. 14.. 4.4. 1100 $1.080! 3.4 h\.bt.1\: 4.5.0.74 ..14 #014 . I 4.. 3100. .0. ......o ..0 l...0 .I. 30 o . . I. 131‘. ....9 -I: ... ......va III 4 -II. .. .- ... .0 .4 . . . .. ., . . . 4.0.9.. 44., . o -. . $001.21.. 110.404. .3“ U1 4...." 740.11.: nun-40.0 .8—‘.. ....4 a ..0-...0. 1..I4fi4 ...‘4 .2. 14.40. 402.3% 010.14.. ...-0" . 3‘. .30 01.0 0.440001. 44 ... ...000-0 04 .0 5:90....” . .04 0,4 (- .044... 00.01 -0 0- 4. L0 4... I. 1 {.04. ,.Lfl-I 1.10.? .0. ,1-44 . .... 0....0 30.. 04.0.. ...-410.. i“. $0.010.- - £6.04 ...-M30114 4 I. 2044. A4. .. ... 3.4. 30......1..- .. 830.00. 420-... ....II 4 .. ,44.,.4:.. 01.4.8.1 . o0... .. .44.. . . .. .4. ... 1...-.4... . .I.}..t... ...0 I I . . .2 41-. . ...0...- - 0-0 1.41-1.34. 1...... 4.21 14.... ..\.4 0110:1011 .1404) . . 1. .3- 401”. .. 4 I}; .0. ... 0 .f 4. _ 4...... .I.. ... "..010n033430-30 . J .4 ......1 .4111. 341544.07 .4.-1:. .. ..0. .2 I .4 40440.. 1. . .. . .I. 0-1.. .4. .... . .. 4. .... .I.: .... 9.321. ...0..\ :4 1..-I334 II. .4 44.1 .3. 9w ml. . 5-9501? 4. 0-. I 1.. ,v 0. '13.... 0 ." 4h? 00 .4. VII I 0 100- 0, 0001. ‘44.- . '30- 40.. 0-0. 0 1 .01 .00>.- 0 . 4. 4 .0. 104.2001... 40- 0.30.1.0.‘ 4:0 ..,. 40 . . . . l0 . 4 4 00—11... 0 IO 0-. 0 . LII. 0004-0 0.0.0 .4. .4 1.044.040- .1 1.... 000-2..- 000 .4. 4.... 5034 414.410....4..00.«u#04.1 091...... It ...30’04014)... 54. V .I.-41 4.0.. .. .o . 1.34.14 0. .19.. ...-40.... , .... .0... 3...,..!- ..0..1.I.l! 4 6- ...0! .1 . .. . , . . ...- . -., 1.4.0... ... 4 7...... .4... .. 004. . a0 aha-.I....”I 111......041421104 ..4. .Idra ..0..- .l 4. 14.0... 00 0.. 0i..h1..8.3. 4.0.0.011118030. 4:... 0 300. “1.0.1.1?! .... .I.. 10.. «1... I .I.... .- ....1148. . ... 4.1. 2.6.4.4... .... 0-.93410 4.1-.I .25.. .. fl. 4 34- I30... ...0... 4 44.21.. .l ,. .0.4....n.1.3 i 1.40414. .10.. lb 9‘.441L..- .10 :14... “.2..! 104.04....- I. . 441...... ..0-5114.01.44.38.‘ I. B. 7.44420..4 .14. 444.: 22.2w“... I . ‘0...’Or0...8-44144I4..-‘ .4. 3.0.9.. 4.. 04.00-01.- 41.1.1443...) ... out 4... . 4.0.01 0 . ...t. , N10 9.1.5.3.. 1..-4’s, ...0-2‘ 14. 2.8 4 ..0, 0 40. 4404134 3.1.4.,- 0004’ 47.. 0- I 1.3140 34.04.41.000“ -00, ,4 ..0 4 .0 ...I ..0 40... ......01 «I... II.0.,- .I’ o 3' 4004 1011...! .4- 8.101 3... I130 .... .I. 0.00. .00. .. II. -.4 . ... .12 . .. m. 0,-04.I.04 I. . ..,-10. .1000... .4 ... :8. 1.3012433! .40 4344.00.51. - ...4 4. .. 18101.... .DVI 4- I5Vh0dw .u .4... .04.. .0 0.! .I.. 4.14- 1. .... .4 z .- 31.... .....91. .|.I .0. 1.06. ... I r . ... . 0014......14.) . .. ...-0131... c . ,. (4......34. {...-10:1...- .I0...¢.13.4..4_. 3.01.11.40.43... .4. , 4440......4...l4-014.001.43.10; ll. ...Rohnfl.‘ .11..«04.¢\..4 005340 I .I-It—u- 06. at. 1.. .111... ...0-0.1.... 3‘ .. «... .. .11... ,.0. III-I0 4 4. 1 3.31.0391 .1 .. ..0 30.4 1.8.!u..4. ,-0|: . 44. . .4.. ...-.0 ... ..0-3......4 11......- I5. I. $041 040 IP14 .4- 141$}...I- . . “a .0 .I.. 119.1% 2.00.. $441.41. 32‘... ...lr... 1...-11... u .41 .- ...043040H4nu .4 0.2.0.4 .331. 4 .04. . .11. .I.. 5 . .4....Y . .....1 .4. 0-. 4.40 .4... :4...- ... 0.0.0- ..0. I .0404- . . 211.112.21... 01.170.549.29104150. $09 . 0.. 0.0... . I .n .0204 d I r 0‘. 0.4.0.. '3 .0 .00',. 0 1‘" 034-009.40.440! .0.0.0.0.040100 :“0 01041 Io. .. I. ... . .. ..0-.0 0.000.- 401-... {4000.41.70.370I ~40. L .>. ..01 4 '43 n, .3303 ‘00. 3‘, ......1 . .4 .00. I 40001 10. 0.00 . .00. .010. 0. 1.4.. -0000. 0 , .0040 000.0 1.011.. 44.. r .4.« 4 . . . ...‘..)4._1... .0.a."r40¢ I.“I1,00 1.0 I 0-, 13.10.41 I ,4 ...0. . 004i 4 ..0-31!. ...0... .9 “.I....3104340639. . 01.000. . . ..I0 ..0-... 01‘ 4.. 0.04. Is 0-1 0. 0- .. .5. .. ..\ .. .- . ..0-.404. . to. lo. ..-...0'31. 1?..011LI‘. .. . 010 «to 000. 00'. I, .. "‘0‘ .11.. wuu'pMMk 0. ' 10.. 0| 4. 000. I . I 0 ”‘39.: 40040.0. ..0 51.0 I .I’ 0 ...-31 0. 1.0010 3.14004... . I... 0.. ..... .. I . I... 04 .4‘ .0 I. 1. .. 4 .0 0.4 0 .0. 40. 0...,‘7- ‘05“. . 0.0“00..'000J11I-0¢0 .. .I.|fl..04...fi...x: 00-4 00. .0 l- > ,, (.01... I. .. 241». . “0494:2404 #:0311401:- 540. d!>x.,1...oo.02 .1- 1...-40.1! ...... «..0 , 0‘. 00.1.!114.1..f 138.3.) «I... . . . 111...».4 . .. . .. . 04..., .4. 0130507130911 .1351} I) L . .13. £41.11 .... 51...] 44.81.... . .030 1 4 .4 0:2. .. . ...: - I .4 ...!‘4- ...0." “-4.10% - -1 .0110 0.00... 4,11. 4. . 4‘4. ., . I 444.. .410 2 4 00 . I147.....1.04:... . In .15 v ..0..( .. I .- 0. .4. 0404., 4.7... .I..) .I.... I........4.4.ou41 34.5034343134 04.4.4 4-9... 41.14-,.I4.0.‘~.-44.N 4.44.01.01.0u}. 4- 0. ...‘1 Ian-00.34.1N040 ...“.9-1 .0 .404, I 40.. 4 .4-140..._..044 10.... a .0410: . 3 . .I.-4 d .I.}! ..0-.001704‘0 0.0. 4 ..400. «0. .- . .. 4 ...-.0. .m- I. 0401.....2101. 4.:3 1... .400. .0404. I. . 44. .....- ..Ha...1 . I 04. . ..o ..f‘.....w,pthU.-.311 4 I ..0 15......6121L: . 44 .342. 1.. .. I. 4 hi...00.1!10.0.2000.n4.400 .2..: I... M .0..1.,.u1. 40.40... ..9 III..- ..4.. 40. .010. 0- 3.-.... T .I- ....K-vasl ...-50.1. 0 0. 0 .. 4.24. I. . . . (4., . av. 413.0043... .I.-04‘ 4 I 44.0.04. - 04-. . I. I. ..40 (..03‘0553 40.... .00.. I. ”71444.4..0 .- .... .. . . 1 2 .4. .411. I... 4 . . ...-43!. .3414 I... ..0 440. watt-r 1.4.0.. 1.. J ... 1..-00$ .. 3....-. . .I.. . 34.4. .101... - (.1. . I u..- 4,0. 4...- 0... 1 . ‘40. 0.10140... 00, ..II... - 15131131 £400... 10-11 . 0... ..,.l. 0). .40 .0 .. ...-.I....Jmuc 0.4.10“ 54'0“.— .50.. I. a ”$4000 I u. 450.44 or. I- 0.3:- 3 .I.-0.4414... ,n .. ,.4..,..n014 ...n". .0 , ...0-.0. ...: .I.... .. 14-0 $10.00-uau1; .. ,- .I . . 0. ...4'4 44 004., v .104. . «4. 0-0- 5-9000. 4. ..l . II. 14.4%.) .14‘0430f: 014......1 . 8g 4..- 0 4H . .. .05..- ....fiolfié... 07330.1 .4. .- ..... ..,. M4. 0. :4 It 0.... . .o \ 4 : 1.0.4... .0 $14.11 ... 0-10.70. .14.... ... Ik: .. .. .’(.~4I...4 .4 I 44.. .64. .1. 1... 1.0 ..., 4.05.01.11. 40.10. .I 41.4 . . ..I. .0: ...... .u... ....- . 4... I1 - 4 144.11.. 0-.4l0.4 “I .11....10 ..1 4 . . . 04 ‘0- .of. 1101.40... ... I. . 4.40.10... 4.4..- ..0. 1*... 0-33. . 90" . .2. 04.: .. I. ... ..r0 . 03:00...” 4.... ...0 1 0. -. n . .14. .44.... . .1116... 40... 1.0...IQI-4-40 . .0 4.“... . .4. .. ... 4 >1. 0. 4. 44.46.4- . ......oi. I4.4-1no.0 211110.. 4 .sr0‘io .- 50? ...-..0 “...“..N... . v 3 41.3.4... I- ; . ”2:1 4. ...00u2-Ii .10, ....4 1. I: o .10....’.. T423. . v24... 1443.40.14: 31,- _ ’..2...4o4.....030.14¥1 . .....l 4... 107.0! I. 0 ...4. 11414:... 4.00... . 0.1 u 4 . A4413 .1 .44. - 90.0 00. 4‘. . .'.0.. 0.00.. 4. ... 24- ,.l .11.! . 8.4.1.90. .00“! 43:40.; ‘4 . 4 . 44.4 .0444 $00.51.. 4-3...‘”.. 0.0.4.048 ..,}...I . .... 1.3..‘11.!L. ..0-4......0013 .0 1 ...III. 0.0.. .....141..«....... . . ... {0 . 0.41.44... ...0-34.1410. . x .0141 ..4 .4.: 01-01% . . 0.04- 4 L.“ ...0. 3.. 42.10%. 11.4.»...4n-‘L £013.44; 2‘- .. .w. 054.8401 .I.-1‘40. I 1300003041,... .00.... .- .-. .... . . .3445... “.00. I .0..I4. -.. ‘34.. ... 6, .....IN- I... 4.- 4 . 4.0 0 40.4 .0. .I.... (..0-A01; .0.- . 2 . 4 ., .0 - .0V 4 .,II 4-4. . 04.0.. L. 0.1.3.7441044 ... 4.0.4.1130. 10- 14 - .. , 1%.. .1. .a . ...-3.30.1700. .0... .4»... tau-...... .1.) 4 . 4,0. .4... . . .s .. 40.414043 . 1 .... ..l.. - 9.404 4. 40 3......94... .( .0. 3- 8 ...... ...0... 4... ... . 4. .- .... ...l. . .4.-10... . 24,00... .1 44. 4.... .4111! . . . 0 ...-...0 .00.!1,.(->..40.n41.-‘.30 '13 $.1ziioh10u41oc01hm01 In] . u v- u. 4 4. ..L..‘ .hy..!~400.’.04r81avl. . 934 .4 10 .40....31,.Y. 3004064313 .4.. ...-”.0“. .800 .1 :- ..4.4I . 1.3349. 48.7 . r0: . . 21111. . ...0. ..4V . 2 .. 4.. .11.. .4. 10.. 1.1. ...II... .~.4.u..0 4.44 .. .. . ... .. o . . .1094}... . .31.. .r.'_40..,4. .4... 34404.10... 11.24.411.041: 031200.40. 4.4011 b) . .4 .0 .Io.««.. .. w. 00 ”pom. .I.-w"... I 1 4" 4 ‘ .m-o‘l.‘.0 . 4 4 . I . ...-.0010. ... 00. 04.041441! -.. I022... I. 1001-. 4 0,404 .. ..4 In. 0.. ‘IIo 0. .‘0 0.2.4 00- , > 0 4 04 001.. a. 40-0 . 4. . . 5. “.01.. I .0 ‘00. I.!.. 004.10. 1.4. I..Iu0:L100‘403.444-330004.0,4-LY1450“0‘1“0..f}. .. 504.4 4. - .1741: Int... .. I ...4 4;: I003! ...-40. .44. 1...-RI... .404 .14...- l.. I 4.4 .41.. .|l.. .1104... ., .3... 4.. . 1.41. 0.... 4: . . a. .....n .. . .1 . .. . :00. .11).!!! 4 ..0...- .....I.1«.-.04...§. 1..... . ... . .. , . .I.. .4.. .. .I1I,0I....0...‘54I.1«411I1. 0.0.1 .14....00030: .0 . 4 .. x .4410 31”.}! .22.}..1-444101. “'1..- .1..¢..t..3.0ta.44.. 1.. .4 luv-44 .- 9.40... ’ 50.30.314.101... .0 .4- ... . . .... o .. 4:. ..4 .. .08 -I. . ..0 I. .0... 4 ELIE-.00 4 ... .9. ...0 .4.. I. .4 .. .....v- . . .. . . . . .. .4;- v. :34. ..., .4 03.1.14 -00. c.1104.’44101,4041”“4.4. $0.434 .0 , . )4. .. .1 440.3004 4110\402 agate... .- .. .... «...-I. .= 1 . .....231915 ...«Z P94 4.. 4!. - ...-.13... .1334‘10955 . . . . . . . , . . .40.. 0. . . I .. I0... .0 . . .30 L . flmgLfln‘o .Q.00\u01 - .0. . ..0-.00... 0.040 00‘41 . 1‘11...‘ .301. 3-03... 0443.4 0.0.0.0410 -0 00 >0. L. 04.0... . ... , . . . . . I .J 00 7.1.8 I .I.00. . . . ..."... inf... .4..00..4.Iu0... . . . .4 . I: ... I ‘43? l 0.. . .0 I Aux-mu...“ loo r10... .40... . t . ...I.... 44- ...-.4. ..01. r141...4-‘-...4..10.I I 4.1.... l 4... , .404. .. . ... .0 hi1... 0.0 4...... 4 00-. ._ ”4 110 .I- .. .00 4.141 491.01 .o o I} .4.. ..0. 410.2. o. .4. I, . ..-.4 4 .I.! - . nu. .... .- . ‘0‘- I...’ III 0 . 4”““035441m«..3’-II..I.I-hzlwwWIFHng'... ’3: .6? 4?! 40.01.04“. 32.0 4.4 .40. 0.01010” «or. 1 10 0.14.010"! o O. . . . . , 4.0. 0. no .. 1 4 .4. I. 40- .4! . . 4 w 4”! fl l...1w£8«oh\¢nl.l...f. u. ”13.1.40.I ton-.31. ...0 11"....610. ..,-0.1.3.1 .110: $1.41... . .- ... ... ...10 I 3-001..- 4. , . . .. . ......V. 4L4.u..ov.~o1.0 4 ..II. .. . I .- - . 1| ..4 7.1432. .04.. 3) 1.. . 1.040, . . .I- .I .10. ... 111.4 1 s I . .. £44.34 .04.... ”Io-,.IQ. 4.1 no .4 ... V _. .. . _ .. . . , .04. .I.. 01.4. .. . .000 ..0. v1.4.5.2r4341gL. .0 . . 1"3003W. .2..? «60-1-04; 0001+. 0. 01-3000,. p’uo ILI .0441. N000 .1. . I. . ‘1‘: . t 3.0.0.41". N..0010.s?..’4h0..00 0. . . , . . . . . . .u v .0....>L.: 0| L ..I- LII. .011 .4. 0-). .o o 0...». 8.443.011.44; .043..- .i . , 3‘1 «44.4.‘. ..0. .4 4| $10.4... . ..44 .4.... ...". .0 .I ......JI .3100. 1030104.)..- 1'40 1:0... 4 3‘8301020044-748 . .,.00.4.4- ...4 . .0: .0 . I .. 49 1 4.4.... .5... ... 00.0 . ,.w - :4...0404 . .. .0 $00.... 1'.- ..‘4v... .2400“. In. 03- I 0. 0.0.0 Ila-.1010 40-. 0. . . 0 . ...-.10. Q‘3.0.-0. . - .“m .‘II- . .. . . .43. .3. ..0- .. . .fv. .. I4 :4. .1... u ...-0 04 4. .4 for; «.... .0.- .1... 4.. ...... 4 3.00444 .4. 0 I45! . 4903.1. 94.411 1014‘. ..0- 04 19.011514. .0 .4 I40 .4.- ...140040. to .0 -..1 0.40105. .0 .. 4011-. «.1I..I.... ..l0. .I40.>-. «gr... 0 . 44.. .4.-4. ... .4. . 1).. .04 ... .003. . . .. . 2-0.4. . 0:81 0.... 10.43. . .3“... II. ..I . fix. 4 . . ..0-1. 4.. 3.446.. .4... 4f 13.0-52.0... ...0. I ..oJVII. 5.358.}. 0014.... ‘}... ... 40.1 ...- II. - ..0 .. ...0. 53.44.0014 .I. v . .0. ..0 7 4.04... .1201; I. o. . ...... o. I 4».4..4.4-..u .41... 2 . .. .0 a. .04.. . 0... .0!- .. . . . 1.4 I .44. I IV . 1.141.414.41- 1.41.4. 111.131.311.00120-31 4. 1g“ . 0. 014.400.120.41. LL10. 01L. ,.0 LI. ..4 “J. 40 I93... 4 .t 4,. I“... .Jfo. h:f”§§135.:.100. 0.40 I019 ..4304. 0... .3001... 4‘... I I I -.4... I. 000.- - 4. .0 . 4.04... 0‘ 0-. . ..0. o .0: p“ 0.0.”. >1 ......0. ..-0. a. .0" o A [.1 ...n. . ... #2014. .0. ”1.0.4940”. ..0. .7 I1. .0 . .h- ..-.\0. ....I .I.-«41:13:13.1. I... 40.... “0.. I 4.0.0.3410... .4.. 3.4 u .4.. . 4 ...0. 1 o .4900, ...... .9. 04.1 . ......40 .I.... ,. A. . ...0-I4. ... .. 3 $1 ...0-.4... .I.-a... ... ... 0.70.1... ..0 4 14.. ... u- -. . .I I I ,- a... ..I I0.. . . 0... 100.11 ... .1... . .1. II“. 40.1...) 0.4 1.4040 . - .41.... . ...-II): 0.. 10.”. SI‘I ..I .. ..,. 0.4.1: 43:015.... 014.0214 3. . ....043...w4.11402.|.:-:l.1 ... .4. 4 .04 .4. 3... .ll ..4. ...,- .4- .I 7. . . .04 3.4 .54... I . . 0...... c .4... . 1.1.7: 4. 4 ..0:- 0 . .4101 ...-xv .131: .I.-T1... 4.00.044... .40 .. ..,..0: .4!- 9... 40. ...-..0. 44044 . 04141.04. 640.111.40.64. .0..4 - .33.. 1.-.-4 0.004. :0 1 40.0.0 31.010‘50. 7: ..0 110.004 01 .- L-0.~ '13:, I... .04.. 0 .I. I .4 .40. .44 h, 0.4.1.1 00 4.40414 1 ...0-r1414! .00. . .f'04 .0410... .0 1.01 .I .0040 ...-0 I0. 4. .II 0.040 3 . 000. . .I.... 0.04 A. .44 . 4.3004. . . . 072-.010I. ... ,4 3. - 0.1/6.4 u‘ 0‘. 443000.91. 41040000, A.%.&wfl.. «1411.....130. 1.. ..« 0.1-“..0k. 1 ...; ....II .01400.4-. 4401:02. 4.. 4.4..- 41000 .I.... 11.4., .34... 144.343.... .4. ...}.o‘. 40.. 8.4.4.1.»...3 31014 .4031... ... 1. 610.100.}. 4.. .I. - .. .. 4141‘ .I.- ... 0...... . .I.-2:44.183 . I .. ...0 1.....) ,:.J...;.... &J?. $440211... .. 0.4 010......101 .1.P441.1014. .1....1 $.31... 014 41%.). 04 LII-1.3.30.0 433.074! 4. ..0. .4. 140‘ . .x....11010-4ll.... 4.4114144109441111-{90344‘ 1.424.. .I .2 ...,4 .-. . . 9... 18.10..- 114. . . 3.1043,... .04 I44. , - . .. -. ...-.204. ... ...... . .. .. .... - «- ... .5430: ...-(4.4.1.44 II III I I131! ‘08.... )2 w.0.0.~|.10.-I.._ 4. I .r-I . . 0. .4414. .. .4... 200.4 0... .400 ‘4 .JJ ., 4.4;? 0...... .40. ..0. ,9. .... 4.. ’0I-;4¢.> 0009344372130."- ..04 4. 124.000.9100 . 0.4. . . ...I 3.0. ...0-10....5- ... .00...*“-0...Ik-M.-.0.£$14u.. ..u o ..0. - . VII (.... P» 4 . . . .I.. . 4...: .I.-1..... .71.... .01 ... .. 41...... . .41.. ...0400 4.0....- 04... I? ...-{4‘}! 14.4.4111. 1.- 501.. .000 .. F If” ...:z. 0 ”kn-C 0‘ I on. I . 0., .0.u .4. .01-.. ..0-04;." Ir. :1. ... 11.0. .. 3 1.001430... .0 n 01". 0'- 01003 ....II. .4.-101.}? . . I '4. 4 .I .. . 4.44. .I. 00 II .000-‘4... . ..0- P4.-. - ,.- . . I4... I .4 n K. I. I. .4 . as. .4 ,.0. 223.40.03.9‘ I30 I... 40.1. . . I. 4d...:........‘.. 34040 .10]. Sr 3.4.“. I: .0 I 4 I.\fi|101..10.. . .3343... 40..., .... Z ... ...0-04...... «No.1... 44-444.. . 1...} (4-00.. .III..« 4.004. 3.04.0 . 0.1 4 . 09.107.41.31... .- .....oo. .0 4...... 4 0 0:94... I. ...-4.01. - 4- 1 .1: 4 ..0-00 ....04. . 41 14.50.01. -. 40-0 I. ...-1.... .... . .L. .4... >24? _ o. .- . 5140.. .1 .4- .0. I4 - .... .041. ...i ...0 .I. 0- . I. 0.- 1.02.42.431. 4.... .19“ -..-I I. . 1. ... . ..0-00.... . 0-01,.1 » 034.... I. I .3120}; 04... I. n 1310...}, 31 . XIII-4.: . .4.-.0. - . . I. ..0: . . ., ...0... 0 :- .., 41144. 1 ..4 4SI. ..0. 3,9..3r00VIL“ -I 0.0 ...00. 4. .100 .. 10... IL. .0. N3). 0 0.4.. .4 C 1.. .... 3:.)- 0... 4:340. 00.. 040. .... .- [40... II. . ....4. .. .,.._0.4w «..0. .44il..4010.alo 4. . ..4.‘ -. .1. I .. .044. .44 1. .. 0.9- .41... .I ....0 4 4 . . 2 0. - .0 2.0- ..n. ,4 .4. .00.: .0. .4 ..0- -.... I 1..-4 .3030... 10.4. 0.14 .-.; . ..0. I60 ...; . .\. o .4 ..00. 4 .1 .1._00.m... 030 0.. 4. 0. ......) 4 8! 0.11.. .4. 1.4 I .....010..: 1..-0.1.1.4... s ..4. 4.04. .3 3.4440. 0. I. 0-. . 4 .. ...: 1 I. ... 1..... ... 4. 1.1.15.3...130- 4.... :04... . 4.-4 .. ... - I. I .. 01.0.0. .I.. .4.-4 . . 4. ..0. If. 5.4.0.190. ...-4 ..u. 0410:44- ... I 4.. 70.1.. . .II. . .34...44...0. 0 L .0. I ..0- .1021; .7 0.0 a." .0... .4 4.0100410 34410.1... .4 ..II. 1 .... 4‘ I... 4.11.1440 4 ,5! .I.-01. III-IL. . . .I.. v. .14. .4. o. . . . 4 . ,. - ..II-I. $0.. . . . w“ . (.0 .3... . r. .140 .. ...... l .40.“..4. I _ ...-u .-.0. a... 130....- I .040-4.a100 ...0-.0- .I 0 .... I I 0.0 ..,-1.1.32.0..1. 3n...\ .. .. . 4 .1010... 0.0.0., 4.. .93: .1 . 2041.00... ,. . . u-III..0..4(’ 01. $0.4..43410. 24.4 . 1..-.4."..“44... 00:034. ..,..013043I .I..0 444.1101... .03.. 410.4.......0..40.0.04,\44-1II...0.1‘444...-1?.,u.......1..4-:..... ,1 , $15.44... bi. .4 . (404-. .. .... .0 4.00.44..x.. -.0I .....m4.0 1...-.... 0J4.43.... 1. .4: 311.124.. ... .. 4-0.. 3-9 .0. 401.. .4 ,,..4 4.0. 4.401. 3.1.0.44 .I.... .. 4. . ...!- 1..-.4.: 34411 .040 . I... . 4.4 I 4. I6 $040.10.- 01. .- - f . .. .0 I .3 .0... .0000 . 00;. .... ..0. .... p. 24-33;; 1.. 4| ”0.3.01.1... .43.... 4. 014 .4105. Ina... .0 0.00044 04.... .I.. .041 . .4.... ..,-11.23.020.40: ..lbo .0. 10- 9-0.... ..10 4 . .. .4.. .10 41'. . a 1. ..0. 0. v1.. I). .0 .... ..,-. >0. 1 14 {.40 14.00 4 00. 0‘0 I q; 0. .004. 10:13.01}: 0....- .VI... 4. L101. . 040.3 .’a 1 00...! 4,: ‘42- pnr..0. 0 44.104.00.01. .0. . 00’. I 0 ...0 . -. . 3-74 .30. 0. 34L“)... II .2014 ...-.41“ If ...? 2,140.... . 0. “004... .0 ,I 4.01...4 II.-1..-.t..01 I... .-.4......-. ... ...-44.. 1...-14.3.1.0 5.1- .1314... 4.4.........I.0-4.4.o.,:.4-. 1...-... ...0: (“2". II. .3444 I)... ..40 41 ..403- 1....Io:.4.4.. ,.4. 3.440;.- 4406‘...o40 .P. .. ...-10.1.0.4. .I.. ..104 gig.- 04...J.410.....:,14.1 0 . ...4... 21 4024.11.43.11 . .0. £00... . . .., .I . 3.4.0.4211. . ..0; 0 44044-4... 20.4....0134. .- r4044. 4.1...440120 . .5424?» 4 ......x...441 IIIJqVI. 00...... l0...|.I.-...‘,.I ... ... .I.... 3... .....I II-I4.4I4 . .I. 41.. II .. 41010:.){404' 4 0 4. 0'11, 44 ‘1. 1.4.0400. 4 0.010.421. 0..- ..1 01.0I0... ... .0 30100000410. 0. . 04 .0. n. .0031. .0 l. . - 4.000.. I PhaJtCI. ...0 50.01,":«04Vfi00‘ 0.0.0 12 441,.«0. .. ...\ . 440. 4 ... urLL-‘o: 0004I0 11.‘004J€.-.0.s.‘4.0uu-O .0. 5.10. a. .. . 04.“.- .I .4 .00. . . I I “no. . ..040 . . .040 :3."- - I. «14:. 4-2.0. J. t-oulo..+4q. Isa-.1. .n ...». 1.. 4 .l ...4. 790-34. 40 .1430! 1 4,121.35, ... 02.10. I’ 413...... I :4. 8 44.6.1. I...“ .3. 4. 4 4.- 0. .I.. "I . 40. 0.- .. . .1 . . 424...}... ...-30:0. . I. '0 o :- ‘04-)01.’ I4 ’ 490 .I.-0‘. . 1.4.0:. u "0.00 .01... "“0“”... \.0..00\0...00'>0 0.0 1.50.0.1. 0-0 4.0.0.,20 \ I 0 .000 . I I... 10. :- 040. 04.. . . 00‘.. .000... I. . . .4. 40. 04.0 ‘4.- . J. ...«o... fnfl: 4094.14 .4... 0 3'. o .4. 0.1. .40 44.4.1001, 0., .. 0.4.4.430“... 3.7003}. .4010! . .0144 . . .0 .I.... .I.. .4 24.... 0.04 no I>.0.. ..0. .0 "I1. .. ....- . .0 0I...H.. 000"}. ...0. .. - I10 .1. Kb. III . ... II .90 04.0! ... . 0 4 .4.. .4 44..‘44.I .- o 4000 .. 4.00 0 144. 4.0.0 0.) .1123: 6.004.: 44 ..0..00.4 0. I I... II... . . 0.1.1.11- 00 I V I ...! -0-..0 .34 1 .. . 40-! ...-0 0.. .. 11. 40 .. .I. .. ..II 1...... I. ...10J lua..1 .. 0 I 4.20.3414! «3.7.3.... - 0.041,) ....I. .. ... .. n .47.... 41.00.143.24. 1. ll- ...71 {.1310. I? u .. I4 .44 . €23.44. .I- ..0. 0.. 44401.1... . I .Ial. .I.. . 3,. 04.404 . ..0... I - .4.. ... ..0 ..., 1:1 I. 4. . 1.4....0. ....1l. 2... . 4. . ‘Iz ... ..,-1} II- 30.03. I I Lat-0 000 or 0.;04- Ir 4. 0. . 3.0 .0... (4043.01 .0030 04 . 04 .3 04000.. {040001. .00 4.0- . 0. also 4 .. 0- .00. .. . I 1 04. ‘9. I40 I. A. 41 I .431. .4004 .I. . ..0 1.4.4.44... 0. .0. I. . . I . . . .I.. ..I 0.4.01.0}... ..4 I . . I... I. .4 I J- 4 3.403.... 4.1.407......404-4T....!3.00...0 ...4...’ 4 Ida-3:41.40 .. .41. 0..1!|400. . 1.. .1431. [7.0.0. 3.1.0 .4 .... .I.-.2..... 14.04. P... . .. . 01... “VI. 4. ...... 3 .o It". ... . . .I. ......4.4..... .4. I... I . . .. .o .. . , . ,. .. . 4|...,.KV1!. .I.... ....4 .1. 4W . It. 0,.«44. . 2 - 0. .... .2..-(.4.. 4.. -. v I! .. 0.... .. . $3101...) .4 .«10 .I..... 423.430.). . \....J.... .... ., ,. . .. 4-4 1., - .4. ...0-09- .03.... .0-I.4-?I..4: , .4- 4I. o . ... 0... I. : ... . I ..0- ..z. .4....) II.» : . . . .... 3.3-... .41 .110- 06304 .I.-4.4.. 4. 1..... 1:11.}; _ 1 , .I.... .. 1...-0.940JHHH10-0 «...: ..\l 4-.00 ’0 . . 10.0...v-14. 33'. .I...41-1410!000L1,I.. 1..-1. . 4-. 3-. .440 ...-.40 80.0L014I . .. 10.04. .-.. 4 0.0 .444. ”...0... r0 .... .,.. . , ... 4-0 .0. . J“ A 1.0.. $03,). .431 0-40.00 IIJ 0'. ..0.-04d. 40,11,200... ..0)? 204:2 0.1 3 00:.11- 0... .10.. 4.. .0. I . ,0 0.1.1.... “1..-...? 4 .1110 I -..-041-1.:.,.:0 4. 40040409 000.420.5004 .7. 124.174.03.44. - ., . , . . . . .v ... .0.>4. . 000.1. 4. r I. .0 0 0 7.04104 . . _ . I .4.. 0.. Il“ .04 4 ..0: .I.... 0.. 04 .o o . 4 I. II»... .I.. . 00 ...-o 04....‘0-1.’ , a. 31,00... .1». 009 ..1 02.4 u 00 .4.. o. . .04....04404! boa-... - 4-.1 J. 04... o ... 0.0 4 . I 4. 4. . . . . . .\4.04 .751 .9 . ..0. 0| ...-.1“ f}...l"%.44c. 4.1.0 I... I. ‘r.!... 54044.8...00. .000 ...-0.00 .. .I. $013.20 .1... a. 10. .444. a... .k‘l‘.‘ a 0 . II .009. .4‘0. .1. a“. 00 .10.:1044.) Jo... .u. .. 0-0407.- 444. a. .1 d . 0, 4. 04.0 .I.‘ .. . .03....110-10. - . . - 4 It. . ....n-. . ... 4. - 0. .. .-4« “.3... I .9... ... ...-Lt ( .40 ...-.10.. ...0 4w. 1 “5.1.14.1. ..IC..... 4. ...-I... .. 0 ,.l 0.4... ...-.1401. . . ..I1? 04 4. .4 «. . . . 4. . - 4. . .4... 3| .0- V .0 .0 ... . ..V.-P...: . .4.. I .. I 4 ..0... 4-0:...140‘ ... .0 . - 1.. 9.14.0133... ...-.... ..., I...I4.I.4.....0012.. . 00.1: ...0..- 3‘ . 04.....8. ...0... . .I.? 1..-...! .I. .1 4 .....u 0 00. 9.... . .0... 44w-1L4PffiiliI .. ,0. 41.40.3020 L40 .. v.0 .48....204 00'. ...-704..- 1....-.-44.11fi 0. "ah-41.4! .4\I,§w 000.4000 0 ...-Inneno 4 0..- . .. Jun. 04. “04.1.24 004.1,“, .240“... l I .4- 414... - . 00 I ...- .I 13....- .- 0... .0... ... .. . II .3. 0 10144.1..‘0 '0 .01". . .... .1." .0 3... "I 144.....- I.01.-4..4’t. of...) ... .40 0.. J 4.2.. V‘ >304...140.41...12 I ,.0 .... .7214!)- 0. ...-0. 00...... .4 I .4.!A.....II.,-44 .. .... . .I.-2 413. .I.-I 1..-..0. .. . 4 14.4 ...0...I 0 .n... . ... . ..0 . 010-4 .4 0...... .1 41". .00 ..0. 3...”. « .I 4. 01 ‘04- ...“VI .4. 4. .0 Ova-I 0.54.4... .II. ...». .4 .0 4" o.- 0 .4140. I 0. 4- 4 ‘10..-0‘4o..I0 I0. .15.. -‘ .0;- I 0. 09!... . I-an‘. . v. ...0... LI: o 40010.0 -’ 040 .7040 00 . r .4. 4 ... . . - . . . n. ..1: 4.010... . .0. . ...-4:0! 0. 0.1.4101. ..,..I > 1.14:! . I. ..,". .0 J-1 .7 ... .0". 004.... 34.041, ,.4.4 - ...-‘4 . .1‘0 .I ..u .7 :04 . ...010...4.0-0. 0. .0 ..0 . I .4 4 :11101'04.I..400 ...-l) 10-. .0. .1 00- . .4.-Iv... I. .0. 4. I I4.. . .00.. .4000 .I.. .. . .. . . . 0.04: . 1 . 1.4.1.3.}. 10,1,- 14..-I .14! II 1 I. ...Ké1. 0.004 .4.. (“:03 .. J 8.0,. 0-..(0... .11. I .. ... .4: .I.. ... ... . .. 04.0.- .DIV 3.114.163- I . . .I 4090.. o) 11. .. ,1 1.4. . . ...-4.24:4. ..0- . . -. I . 4K 4. 1 1.3.8.. 4. . . 1... 0. .4.-10...... ... i "I". .1094“? '1..\ .4. 4003.....00447..II.-0I II .I .41. ..4 ...0 .10. '4 .... ..01. .1 no.0! 04.4... .04. I1... .. .04... I....bI.l.-.... low-....IXI‘.00.00 40-40 I. o..4,. - 0:0. ...0....-. :01]... .. . ., . . . o .. ”Wk-.1“. «.04. 12,1730 -..0.I. .... $-01”: 4.40 4110.. ......313... 4.3.41.1... .43.: .. ...3 . a 4 .4! . ..0... 4 ..V . I II . ... .I.. .. I .I.-.I- ... ..00 1 81.0 3.. . .I......‘. ..x- .. ... 34—)... 1,. .. . . . .11. 4:. . . . .«1. ...-is? u. .1 0.0-... . .I.. ..A. .... ,... I100 .00... .I- . ... 710.. . :- .I:.II . 34.0.0 .4. .I.! 5440.3.rt; .... .o.\ . t {3... 44 . 0-.07 . . .4 I 4:! . .. 41 I4 .- 0 4 . . , . .44). 13" 4.11. 4.0 - ...-...... 03/... 0.1).? 4 . o .00 0 .4‘ . 440- If 00!... 0.400 I 4 H 1.4 II .44., 41.4, . . . .0 .00.. 14:19.410. I. 000 4 44. .I . . A 0 . 4 '51,.NM . ... - - 4. . .51.. 0.... I04 .0 no. 0.. 4. o 4. V . . , . . 0,1 .0 00 I: ..0-0‘1“‘1-....-0.! 0V0. 1444 ....0114 u. 4.1.1. .. . ... l : .04... ...-.I.... . 04.3.1- I11..0z-I-.I..I.|.. “.4....1CII. .00..4.l..4. 3.1... It... . .4 .I.... ..0. . . .., 32-: 10.0 I. . .II . 1:10... ... 4.. .....0. . .2219 :4 \4 4. .4 I .1: . . ...: . ...I. ..0.. V. . , . . .30 .0444 4 .140- 1313. 404. .1404 . 1. 5.3044}... 0‘. n ..J. 3 a 4.4 _- .0 41.5. .4 3.0 - 4.1.4.... ., 1400.13... II. 3.4.“ 2.7.3: .00.... I- . 0.44.. 0.. 4 -. £13101. . 4. 451.4. .... ...... 43.4.5494 034.4... .. . ...0 44.0. .3... I: I. .2..... 44.00:... .0203! .3 .4.. 13.0. . ... 8 1...-:1 . . .. 4. £43.01 0.4. ... o I. ..- 1..-...}... .. ,. .0- Lid-14.3.4044. 40.. -..II.. 1.1”. «IA-"‘0... m0...r. 0. v‘«. r9010. 4.. .04 _A 031. 0.000 .0 0.0 .. I; I0 0.4060 4, 0.0000,...)40 . 70:04 ...-.I.. I 1...-3.000 0‘ 00.04 I... 0 J 1,0 0.0.0l .I..” if" :0 u .4. .00. - ..0.’|.1-.‘. 0000 . 4 .. .40- 01.50.04 0.0;... 10400 .10. 1| 4 00.0]. ..,...00. ..Il-.4...,..0 . .0.’ ..0- . .01 4.. .0000. . . .’ '- .- ...0 ,3 0.1.0.44) 0-0... . . .4- '0’Q4~.II. 1“. 1‘04"04g"4 asu= 04; r. 4.0 . 0 p; . sis-.I....Y—LIIIZI- I 4.1.4.. ”:44 .. La. ... ... .If. .- ... . . . , ...- I-a...~ 1..-.151. . «,ll. 01.- ...0 3.:- 414 . 1 L034. . ,0, 3.1.0:-.. .0 ..00 o ....10. ...-0|. .0... 1.. A 4-4.\ I I .I-.0I fl‘lic-Io- I . ., . . .34 ., . ...0 4. L04 4W,0.-I1- ..i’l’n’." ..0.- I 4. .14’. ... ....n'u. 48,.“50CFWW141 .11.. 00. 0:11.744 0.5.1 .4 .1713”. 0....- ..10. .....I‘ I. . .4 . 40. .3... II .1 0.0044... 1.1. - .II.. .11..!. .....4..I.. 17-1 . .1, ..0}. .I .. .. . . ‘14 f 14.0 1.0.. .. . . ... . ,.0-1.3.0.4. ......4- . J. 4 4.0.0 0.1.0.1. ....1 0-30)] x”: I. ,I .4 00.0 .«I 2004-1400.. 24.0.4.1. Va 0. 0. ..>.- I (0 n 4. It“ .. 44:04.03... 0.0.2.00 . I I. I I 44-: 3.043.4I...1.« .I.-nub I. 40.0.- .0 031.0... .001 0-. .0 . 4 ......0’ . 4 00 4. . .. i .0 . . . . . . £40.. 3040107..- 00‘ . .00 0 . 2 . 0!...- ...I . ....4 10:... a on. 0' . 1. ..0 4 0. 0. ... .I.)... .4 Iva-1.00 I 0.0 0.4144 . . .u .4. v.0 .0 I I -. 0. .- .. I 0.04 0' NI. 0- . v 1.0.0 Q .L. 1.00 0-.... . .Ir o. 0...... ..0 044: 40.00000. . . . 4 .. .I.»: - .I .00.! .01....40; 40..” 0.. .. .- .0 a 0. >1... .0... .. 40440 .... 94.4.. r... .00). .I .. ,. .10.. .. 4 .0 0n d034a12- ..4 ‘4... . 04. .....0... .2. .4.. 3 cl. #04-14003.-. 0 0- 0-140. .1721 - ...4. 0.. I . o. . 4.. I .. .0 .04.... 000. I... .. 4?. - . . . . 1 4 . .1001. ... 1 .. . . .. . .I- . . . . . .000“: 42.71.. . .... 4 m «I “LL. 4.833... .44.. . 4.?471-0101923 .! .1 .... 4,- . .. , . .14; ...-v ...; .01.. ... 4.. . .. .I.-... I .. . .I.... .4 .0. .0 .211... .. . .....4 I .. 4. . .I . .. v ..4. ..Iov4 .4.-0 . II 0 . . 44-. ... ....I. o ., , . .. . ,.,.-... 101.. I4. I . 4 .. I.\ 4 . V 0 o. .. .1. A. .. .«l ...lL.. d. ...0} u o. 0.0... .4 cnovo..41>0 4.0-.4i .4 . . 1 01 .044401-1.010... . .. ... .. I1 4. 1.00. 0-... 3-30 '70 I.. I0. 0. . {I 0 . .I. ..0 . .I.-$031.1 ..1. 0. .4 . . ... - .11333324 . 00.. ..0 0-... I... .0 . 0. 40s .. . . . . . I... 141,... “I... ... 0-...V. 0- ., O- 0 .0440: K 4. 0. .7! 0 I40. 0-40 ‘Iia. 00:0 I .4 L‘I’IZ . .. 4 0 000! 35.44.. ‘0 . .0 ...-.0044. 0-04.11... . L. I4 00000 0-00-40. .I- .. 4 - .....0.‘.10”00”.0 10.3.7 00. 40004 I . 44 .... 4. 0‘. .I.. 4 I .00) 4. .....J.’ I . .. - .IVYr ... .. 40 .... .. .2.. .40 30013044 ...0 (4.41. .... 00. 41.4 1~.r44.r1..f J . . . .. ..40 ‘4 v.8 344 .I. 44 (-4....- .0...4.I- .. 4. I4 .10 1‘0. 4. I...“ a... . 5.10 f y. . ... .., .-. ,04.4...4. 4. IA... .011. .u. - .0- sJ..-1. I n.. V6.1 I - .4291: 01.0444. , 14401.0 0 . O 1. 4-..‘43.’ 1...-4.1.4.4. v... .. ,I 0. .04...V.I u... 00:2 .1 .... LII-'10 I 0'. I. ...-.I. 0023...... 00:11. . ,0 -’>4 ...0‘0 034......3-404. . . L . ...-.03. . .00. 0-0 4 6-. ... 001......41 III" ... 1. ,,.‘d: 0. 41.1.4.5"... 4 o- If 4-. ..-4II 0. . . .4 2‘ . 4 . 3.0.01. . .u . 0.0. .0140. '4. 4-. I. .00z‘0. .40 .1.- NI. .40 0 0.04,. 1:431. 4.. 0001.- .V , g. .. I.I-....0 80>. ...... 4 .s ‘ ... 3:.- .. .04. 0...". 0.4.0 ..0 . .040. I04....4..T.... .4.- .. . I. .1 c. .08! ...0 4...: ,,.. ’0400 .I 414‘!) T. ...20. 10.101.091.031. - -- 4-». I. 44. .. ... ,.. 4 4.43314 0. .. II ...1. ..0. 3.3.... :01. .- . ...--~ 0 . .7 4 .10 ,. ..40. .0 ...0 .. l . ..l... ...-01.7114. . .10.}?! .I.-00.0.. . , .I . 040.00 0 ..0-0%. 4.0;... «3 .43... 0 001.10. (C. ..04 ..I . .I‘. 44. ...? ...0 ...-o. .....0........ 00 r. . .. 0.: 0.14.. 4. 4 .I ..0...) .444. {:0- 0. -94I.>-.. 0n ... c . 0. .u a 3 .4. 11.4)...)- l 1.2.... I. . ,I 41.0... 4- M . . . .11) .0 3,4,1. 1’ . . 40.. “...-l. c . . I1. ..1 ... N... .4 ..-.-0. r . 0.04 9.11.0». 40...? 1410-0 . . . 3.. In“... .40 :0. .....I 300.0034 11- ...: .4.-1.40 V 043...,03... .40. .2 o. . ...-... 0.0 > 40-. . .1241 300-0 . I .11... 0 L. . W a 4 0.0 1..... . . . .. . . I .I. J. J .. v. _ ‘00-‘- 4.1.4.0 I 1.1.1.50 .44‘.I.1l .. .1. ... 4.4 .I. '4. 0.... .0. ..0-L I1 .oY’SX -.| .. s. .4... 40 i! ....3. ., . .4, 440....- I- 4.! .3: .4 i ,,4. J. ...0-......1.04 40 4.034 4.... .01.. ...4? .. .0 . o4 : 4. I41 ._ I1 - 4. .- ... . . . _ . . . . . . ,I....., . ... .. 1400.71.13 .. 41143....)1’4713111. ...-.31.! . ! 11.430004... 1.111.... 40.1.. . 4. 4 ...0 I .1 0041.3- \. 10.4.34- 00).40 :I . .04 I ..,. ...... I 4 4.1.0 . o 34...... ..,..3’11’ .0.) .. 1..-.1. 0 ...14‘.0..11400u.4000uh“0.001. .4... . 0.001....- 4... ..0? , . , . . ,. ... . 140...). .0. 1.. 4 .,... '00, I 00 04.1.1! ...-... 4.4- ..0. 0 . 000 00«.. 0.. Hrnb. .>0 9 1’10... .I 4 .104 0.314 '4 I . |.4.4.I5-..4 ...-.40.». ...-4. Lo... 4.1.0 ~..,41 0.04 . I... O 40.0 0 . 0- - 0 I ,0 ....4 .. . .I - 4. .000. .4. 0.4-. V0 04 4. 0-. 00. 4.. 04..- .4-4 . ..0..h0.......« . .. . . . .. - .. .v.I... I 00.0 .. .130.I.1..0....4444‘VI1. ... .1 I ...44.00. .I.: V. ..0. 400 4.4.0... 0.4 o. .. .4.. .0... 1t I 0‘. .V 11.5.0.0. 44 4' 40. .3000- . , .00. II. 010.24 4.140. .15 . o .110...- .. 0.0 .u. 4 . :0 ...0. .30”: I. I II‘V.‘ . .. 0.. 0. 0. . . . . I 0 JIHJII . ...0‘. 4-11 043...]. . . 4..I|...oI 00.141 «0 1- .00- - ...0 1 2.....- .Co.to“.‘. .44) . -lv. .0 40 v. .I.....-. 4.04.0. 4577-. -...-04... ... I .-4- 0.1.1.40... .0.00...-. 0,1 4.3.0 . . 4' . .10. 1. 4: IL... .1 I... . 3...: 014. .I.-I . 404040 . .3010! 4 400.4 .4 ..0-1 .. ...0 0.9... I'DIO 1 I .I.-10‘ .4. I. II. ..4 . lnfl ‘4 30-50 .. I! 004 ‘- I 0.. . 101. .40 0 1...-Lvflv-IIO.0101.OQI- ...0 ....‘uu . .4 ~ .1 u .011. 4-04.. 4.. . . III. ..0. ...0. I. ’0 .I. ..1,0. I 0. 0. . 4.. .4. .. 00. 00040.8. ...-0‘: .2..-.- 444 .4 1 I .. 14404 .I.-... _ .0.- . 4 . ..0.15.4 ... ...0 («'130 . .. .I.-1.3.14. . . .- .. .. . .-I041. ...; 0.. 71!..03. .I.: .. I. .I. 4..“ .4 -44..<... -. I! 1... ... -. .... ...0. 1.04 . .. 44.... - ... . .0 n .4.-..- .. 3443.10) .04.. .2..-I: 0-3114- 2.0 .441. .. .404”: .... 4.. I ..u43v... .I. .... 48I4.-I.I.4I- . «4.44.4.4 .24... .II I. . I40. .. .10 ..3 . ...0)... . . . 2 .4 I ...-4- 42. 4 . ... . 1..... o I: 3.4 I ..n. ....I _ ..0... .3-42 . 4-3 .4. . . .. - 1.1. 4... ... p .3 .1... . .... 01 ...-I 0. “I 410104;?! JI.’ 04-4 ... ..0-.I..40>... ...0. ...It. . 34...... .Iotlroo'! 4 r 40.... i . - ..0. ..0... .I.}... o 09 .0 . .. .4 .I.. .. ...... 00. 4 4. \4 ...... 0 4 .I.. ..0.’ .00 ... . -. . . . . .. . . 4. . .4 400 .I.?I: I 3..--.. 1 . .. 4.3) ..0. ..0... . . .04 .. .I.. 0. 4.40... ‘10,.o 0 .4 0140. ...- v. I; I I: 4..» J . .13... .}. .5 54.40- .v,.04.. I.4001..‘I.-1 . . 01... .4.. I... I .40. 411 ....I .4 ... 0 II. . 4.04... . . . . L...» 4: - ., I .. v 0.04.400\41.4.0.o240.-0.'4. 2001‘. ’1- Xo...0 I-I..0I\qu,4~. .4. ..~ 0 . ..4‘ ...-{411.4 00.00., .0 A... 0. 4.44 0...! .. ...1- 1 4 0... . .. . ’31:... 204.00.040.11. 00 I0. \0 . 4‘ a .1 . 000.... 4. ..0-.31....1. 0.. ..IV 4.40 03.0.... .. . . .‘r 1.,IP‘x1’f. . _. .....70 . 4. . .v . l $0.... I .4. .4.? .....0. I. 4 . .. 2.40..fi.0\l.lo 1..-04.0.. .I.-sf: ......-.3. . . . ...4.. , .2..-(4.1.... .1 .. .. _. ... . . . ,0: v 1...! s :0. . . ‘3'. ..I :- 0 3-.30 I. 4 I. . 1 .1 . . ... .I I ..I.\ .... .. .I.‘ I ... . IT 00.- I 423}. r- .. I . 00.40.... . . €1.40!!- . 4 u o’... .4.... 0.« .. 0.4 .....I... ... _ ..0-,1 .I ..214.‘4L - . 0044- 4. .4- 1..-44.0... . ...-00 . . 1.9.01.1...- .. 10. . I I c . .0 07... ,444-4- 9 ......4 .49 .o ..0. 4. . .. 4 , .00 ... 4 I 0.....-« .4‘40000.4.. .01 .40 lo.- [I 30 0.1.. .. ..0. .0 .4.. .. .I 4,? .4. . ... .., . .I.... 4.. vi 1. .31. ’41.}..005-1 -4,.I.L..8....1. I ...-04.0.4.4 .431! I....., 4 .I.:- 1... .44. 1.1.... 00.... ... . ... . . . It 442.414.... 01.1.3.2:0... 43.9.41... ,..-V.c 44 :04: .0103}! ..-.54. .I.-.1 ... o 40.. .04.. ... . .4 ...... 3.8. 4. .II... .-.1-.... .04 ..0-.... 14.10.I>.t00. I. V 0.”...- 4010.... I .0. 103001010 I..00"..r00‘. t I...‘II.0 .000 .. .o-‘loOIJ' 00.0.40- ...0. .0 cegaiix .44.. GILT 00-0-.I....-I a .' .0 .' ..0-0.. 0. .0014’9‘004 l- 0 0.4-0.0...40004 0‘41“. ...0 .. .... .15.4.... I. ..I. I0. 0.... I40... . (.0 0.. 1.0... ..11.-l . .I.} 0.4. 30.034.03.204. . .-4«.00.. ....._.04..L.-4004..0 0. v .... '00... ...0 «n II0.40 .. .41.. I C. I .., .I.-4 .44 3 - .4.V10 4 .0. I 444104 . . 0.4 .0)- 1 d-3..n.1 .0 . '0 . I . ... I0 . . 0 I .. I. 4. . 4. ...1000. I o- 4 a .1 J 00... ..I- I .I.-0.0 0 ...-LIL}. .........10. 4 ..I. I $01041). II 4 1’11-4.‘ .ll” , |-.-..I....\.u. 4 _- I... .. 1.44.. .0 . . . ,..,..v.. ..I . {NJ-37.10.... I .. . . .l- I. ..Z. ...-4.31: .I.-... ... 0504... 4410.24. .- . ...0 .4 :- . . ... .4. 040 .. 0 .... . 040. o . I 3.0.0.. 4.1. . .. .I. .4. I .0. 1f. ...-.0. .. , 4| ...0 .. ... . .... ,. 0.013.339? ..0... I. ...I. ,. 1..... .3110 ..,”?0. .10 It... .04... 1.0.,30. ... (1400. .4.. . 804001,.34. .h . .-.0.4, , 4:4... .p. .I ..0 4 .4 - .. I ..0“ l_l..l... .00 .. Io. . ..E ..004. ... 3.0.. 1.. v. .- ‘ 1 . . . ..0 . . .. 4.0.4 . .3 . l .4 ...-:14 .04....I.’ I .. .I .I.... ....I. II .o. 0. I ...0... I‘L'I 0...... ...}..l‘ ... 4.4.413... V 41. .I. .10 00. 41.4044. 0.1.1.401... .41 4040 . n 0 v A. 4 ..FR. «0“ 300.....2’ 4 .. 6.1. , v04 ... J. 4.... ..0, 4: . ...-4n. “.1...- ..0. .. 0| . n. . . .. ... ....If)..04..0-I.. I4 -. 4.04 4 ...I.. . .4.. I. .. 0". ...0 ..0; 1...-43.4.0.— .13 4 ,1 . .. I..I..4-Io. -.... ) ... I. 4 50.0.. W... .0; . ......H,-..-I 4 . ..0. I.-,, - ”...-II... . .I.’. A. 4.500.114}, .1: 00.-00I4.|\ 914 .4770. .4.. .11404041‘0‘41 4 ... 0.. o . .... ...... . .. I c ....4‘... ,.0 4.4 . I. . . «.1444. c . ...J? .. ..L. 0-. .- q. ... .4. 4. ... 4 .... .. i .4431}? I. ... .. _ .. ... .. .. .. . I . 4 4 ... 4. II. 4.. .. a a . ... 14.1013... 1 2. .... 4.. ,. a. .. .4! 0... .... 0-0. 1.4 I. II .. .40 .114...- 11.911.11.140 . ....413fl .I > ‘0 .0“... 4140‘.IK\01..4..J, I0. 4.«n..~0....:.4. 00.90 I .09.. .. .I.. 4... .0‘...." {MI} ..I,:: . 4 00004“ ....«...- .. ..l-«I. .13 44‘ 30.00.:- .1IIL .. I- 5.. -,.. 40- -0 PI... 5. «.I. .443, ,> 40.4.. . 0- II .o .v. .- .03... . a 4513....- . 04- 04.0. 001.) 4. I110 . .. c ... ...4 ...0-“31.. | -. .I. 00.4}- II.0 I. 000-. 1" >010 01003.40111‘44-11‘1‘.» 1. .000 i s I 4.... .... . ... \ ... . .0. .0 9...... - I 2 ..I. a. .. 1.. . I. .... ... .. ..r. . .... c .4. .. . «m X. 3 .0.- .. 2 4. 4.. ... 4... . . 1 10- . 41... ,0I .... . ...4 1.. . .. 1.10%.. ..-. .0. 0.0-0. .13.. 2.4. 1.4.50.1...1 041-2000.. $11-44, 0110 i4. ..fluflI «.1 n 0.... u .. .4 .. . 4. 00 3.1.4.015”. 4.. 404-44.. 40. 3.4 4.. .1 0:4 0 ...-...0“. ..,.. «Ian-:1. ..41. ...... ..-0.o..... .4. .I. .o I 4- u. .. 4. ....0. .0 .I. . I... 0.4... . .| :05, ... .1. ..0 «4 -. .. . .. I00 .. 24.. I 4 1 I... 0 02.0.4 .., I v . A ... .W‘. .| 4 0.. . I I ...-1.001(41-4 ...0..140(I0.00O 02.04.400.01. 01 .I.-1.3:} .- Ilitm .4 rAV 00. >4... .4030; . . .. ... 1.2.1 04.. .4 .....I .I.... 4...; M,0.v.- 044 ..0004 I .. 2-. .0 u .. .04. ...0-0.. 4-. .0126... . .04... ....0..4|0 . .- ..,. 4. ...-.414. 3.23. ...-I ... .04....3. 5:0-1 .... . 0.4.04.1 II.III-.40..v04.. .004... Al I .0! I .40..1074I.:100- . .. .I.... 5.0 04 {>0 f. 501....”- . 10.141... 4.4«10 ..0 9-4”. V . .4 ..I. ... ...-v... 0.. . . .I.. I“. ...-0.... 41 ..4 ..0-0.4. , .. . v. ..- . I... .4440 .. . .. 04. .- 1.50) - .4.. .I.! 0....-...4. {1..-0.20:1...3.‘ .- - 0. .,1II 400 .. 10.1.1100 1..-00.0.5140. ...-.4- .4‘ ...1! £6.31.) . ...4.. 100.41.... .. «hm-1321.5... . 7 . 0.... . ... .7, ..144 l- . ... i .t. . 0.. ....A. I. 4 . .4.). ... . .I. ~.. . .... . .,. - . .. a . . ...- ..05... ,0 :l- .14}- .. . . 4.4... ...-... ...... 0. .~« 14 ...4 I .4......- I. F p078! 4. .2....11... .- Ix .....1-4001’14..060~I.)>ot..4$0 r4010 .. 3:1“, ....I: ..LI 0. 4. 01. . . . ..0...I-0.4' . 10.1.0»... 14.0..0 .. '00... ...0. . . .... 0. . ..4 .. .0. .It..-I.L...4 0 4.40 - . 40. ‘00. ,..- .... . ..0}; 2 I I. . '5 1. ‘04 1441.. . . - ~13... ...0-.010 .301. . O1. 0. . . 010.01.... 4.314 , .39., II, 00010)} J ,8... .05» 4 3‘ II ...0’0 . . .I.-0.2.0145: , 4,. . o. 04 1 04.0. 40.0. 3-. . \IL. .0 . . .304 - ... 0| 4. .. 04‘.- o. ...! 1. ...0 I4. . . 4.. I . .0- 1.0.... 0.. .4 0. . ...0 4X .0. 0 001-0.. 4..I 4-1 ...0 -. 0-1... . 010-},1441 ...-.1114. 40.1-I.0.1040..... 4.344.825I ..f ......14 ,. . .51.. -... 40.4... ...-44 ....1...:4. ...... .. . . . . .I. ....‘.t.14..so. ......14. 8......0. 1.1.... . I... ..l......4¢..- .. ...v ..I,...-. .. 4... 01. . .. 0 . 1.4.I.... .-....l ,\. :21... 111.11.11.0- .44 . .4 .4. 040.... .44 ...0. 4.0-... . .4 -.vI 1.000404...4..l of... V0.0.40.>I-.. ‘4 7.000 0.0.0. -I.vI., 0 as... 4- 0.... . .... . .4 “-0-“ «I... a. ... .. ... ... ... .. 4 04.0. . f... ..0... ...0 .0 | . .. I0... 4 .0... .0... 4.00 . 4 ....A, 0 0.1 .....I . .I0I .1. 4:- 0 .II' .4...0~. 4.. 401.. 04.0.4I ..1 4400‘ 0.0.00.1.- Uzo: . 20.4004 ... 4. l «I .4. v ...-2.2.0 -:..I!.. I o...-I....l.._)4 ... .1..0.:1.3.u44:... .01.... I .0415: . .4 .I .I.. . III-.13... .4.. 4... 0— .I -4. ..0... ... ...... .4.. ...1... .. .. - .... . - .... .. . . .... . ... .. . I- . ..,4. . . . .. .1..1.r..0........0lv .- \ ,.4I04I. .. ...... 0a.... 0 0-... \...0 0. £494 .I.. 02. .... 4 .21 .... . Z 7 .44. -. . 0 . . . , .. .00 ... . . 2 . 16.040... 404 ... u .1 «I .0- ......I 0 I... ,,u .I.-.I.. .... 4.. 0.40.. ....U . . I... .. I ...-4.. . - . .0-0 .,44.1,..I . I. .4.-... :4 ...! I31 400.141.4004.. 0 021. Il.«.1.0«.- 4.... .01. 4'0 000. ,1: 4n..- :. I. .4 ..2, ..0- . l- ¢II3 .. . . 40.4 ..0... .. .....47. .. ..I ..ISEY . 4. 40 .4-4: ..0 .. ..1 V. 0... 14‘. 3.. 2.44.. .- ..0-v.1...- . 1.44 04. .c I .49040. . .414 0.144.. 3401-.‘.. I 4 ... >0 $I.I.-)...0‘.443. 0 .4 1.2040104444101110. ugsltoosopfla. 0 41.4....40 ...-.010... . 4.. . I .4-1- .1 0.. 00.. . . . 4 4 3241.111 .- .4 v 0. IV I, 7.4.4.70... 4. . .. . . , z. .. . ..1 L... 2..- .u. l. .I. ..., 0.- .) I. 0.1 I. t 1 . 3 .1 I .0 I . I VI. . I 0-. 4 .II. on M 4. -l..-...0- .....I4 4-... .I.. .510... ...0 0.1.0 .- ...-0;..10310 108.1 .00. 4. 3. . . .p .4.. , . .l 4 $4.1. .. 41-4.. ... .4....Io. .0 t . III. I I 0.1.7.. . . . . , ,.. . . .. . .4... . c. . . ... .. . . ... .... ...... I 4.... ... .. :0... .. ... 2.1.1, .o I. ”...-... . II 0... .. . .. .... .- .... 10.1.13 . .0. .I..|:I. . 4.. 4. .I! {-0.40 I; I no.4. !!.4-.l-. t14...0411-..11Ii£..I-II. ... 41103.... .I. .. ... II .. . .. ..0 o -.4.- .4.-...... ..Ilg. .10...- I. 2.14.. . .. III... .. . , ._ 1. n . .. . I. o .. ..0 ..... 4.1 .l ... ,,.. 0 4 .. I 004. . .... :04 .. ... 7-1. 0 . 14. ...-iv. -4.. .I .41.. I, ,...9 .04.. y 1 01.... I201 4.04.5.8- ...‘IMI: ..l.-.-1r 04. '- 01. .54 . u...‘ ..0. . .. Z .0... I. . . >... .. 4 0.00,. 00.0.} ...1... . . . o .. I... .I. - 00. v . o 4. . I... 0. l1. . 3-. .0 ... .4 0- ..0....- ..0 .1 o 0. .1 .. 1 I. .- I. .v ... .0 ...)...II-. 0 0A. . 0- I 5.1 .0300... ‘10? .II‘..4.,.II. 11.0.0.0 . .J. I. . .....- ,t. .4.... .4 .. (I. .. .4 .. . ....f. 3:... . .0 o . . . .. .... . .. .... .4...I ... .... ... ,0 I 0. . -, ... . .. ,. - . ... .. 1.401.... . . .. .. 1.41.). , .. 4.. ._ .. . ... . . . ,. . :11. . .. ......I . ..0 . ..o .4- {1.14,Ma’s.-.-441.01.113.153421 I ‘1'. .0. :- . 004.04.; 4 L. .0. I o.1‘...0 74:0 ... 0.0 .4. 4.. 0.4 .0 4 . o 0-0: I0.-|4_0 . . . . . o 00.1.. .. .., 4 4 0 10....- I I. O... 3. 4 . .o .. ... 4,4 9.! o. .I B. ...0-.... L s ...0... I. 4 ..44 .I.. 41. ...1..0., . .I.)? . . . 0 . .I. .I-...q .. 5.0.54.0. 0.111 .0 4.04 0.... 0.. ’0. 00\.: . .. .5. I 4...... - ...... .4 . ...? . -.0 . .... . 4 o. . I I... 42,. ~I.0 . . . . 4? .. oI . t . .. , ..I. - I. .0... . la... . . 3 ...I . . 4 ... .. . ..0. I. ,II. .. _ . 1513,)! .73... ... . .s.t..... II.....10...,| ..0-:4 14..'.I ..-\ 5.1.2.404. 4004.4..(4040017 . .00. .4.... O .. I. 02‘ ,4 LI 00.0 x.. -. .4 . .0040 0. n . I 0-. .47..» I. ..I . ._ .0 4 o 00'. . .. 0-0 . ‘4 .s I. ...: . 4 .I ..1 I. o .. 0‘. I ... 14 I ...0- . .21, 4., 41 4. b.- - 1.0-1. .4. . ...-£40... {4111. . ,. 0.. .4.: [Ln-0 .IHCM. 0-10 .0...‘04.,I.‘.. 0 -.. 4 0.0 o. 0.04 (III .4 ...0 4:1. . .440 0.10. I I 1.4, 1 s 0. 2 . 1 4 4 ..0 . . ....O: 4 0| 4 0 . . ... 2 III .1. .00— .4 1.04 £0 . 1,..‘ I . \0 14 VI 14 .0490 . .I.! (III 40., 20.0 .4.-4.2401010417-0... .4. .... I 100 I 2 n. .1. .... ...0 I.I .I - 00 ... $4. .4 .... 4 I ...... - . II . 01.. . . ...0-.1. - I .. 0.4...I4I4 -... 11 I! 1104.)...- 414.403...." 0.040.041.3511... V. . .x. 00,04 01.0 I I . 4.. ,.V. ...-.I- ‘ -VI0 . .40 .0- l . ’0 4 4 04. 40-10 . .4. 000 .4.!- I ...L.m 00:0. n.00 .. ..I .. 0.. Z p ‘x-- - ., 0- . I. .- 0.. .0411 M0- 0- .I ‘4 AVQ‘LCIIII. I 100- ..0... I4. 4- “ o-COOIIIA r3’2'0. ,2‘441 ...«II 1.1.... 40 . :0. 0:1,. .. .41....0...l-00 144...}..0. I0 .... - . . 4... .. ..l. I! . . 400. .. Iv. 1.4!: 0 .- - II.- .4. . It 140:0.- .. .1. .4 II...I.' :04100-0010 4“. >1 0 4. IIJ0~".. . . 4.00.. 0,41 04.0“.4 ‘4 .. .... 0. , 40. .I. 1..: 4.4! I . . .. . . a... .... .. 111...: ,. 0.. 44 04.: .. .I. 0‘4“. -..0. ..1 0 .- 4. . I I .0 ‘4 . I»... 4 0 4 . 4 0. .244 I ...I 41- 0‘..- ..I 01.40, .0 .1. ’0. . cot-1.434.0’4I‘IIL .I 4.40.. o ... l ., ... . . ...: 0A...“ ... .4 . 3:41... . , .. . u 1..... ., .0- . .1 . . . I .. .. I. 44... c.0410... 043.1;- . .I I...I.- . . .. . o 4 u 4.00 . . I1... . .I a. .. .. 00 .4, 104 III. 3.1-..1 1100,40,..JI I ...: 4. 4.4.4700 .I III; 1. 4:10 .. . . .- .I- . 0.100....lo0- I..~0 I. 0... .,. . I. ... I I . 00-.-. . . . .. .4 .00 ...)... ,.0. ...i .. 0 . I . . . .0104. ... . ...... .0. 0.,L .04 4.04.121, ..‘~40.|L3047 I 44101.02; 0:3..0! . .34.; .0 . nuuorqvcp...4., . . .. ..4- 1.21. I I. 44.0. _. . I ... .0ss .. _ ... o. 2 s .... . . .. .4... .0 .0 . 0- u I .. 1 it - .L- ..0 ..44. .. h; O. ..I... (4.. - . 1 . -.-I04- .LIII‘ . .70 s .4. a 1- Nab-“..QIO- . II 00044. 0 .. ‘0. . fi . 0-0. >1. - . 0-...l.04- .1 II. .... 4 , ... , . 4. 4.. . -.4. . ...)... u 4. Jon] ...... 0:. .I . .0 ..0 I .04 . IO. .1 .4 40.4..04.J 4.1....- 3~II .44...- A ..0? 444.........401.- .0274..- .04....0.....(. . ...... ..., . ... 2.1.. ... .... .. ......I . . . ..1: . .. . - . 1..-.93... ... . .4. 0 444.410 0.. so 0- I. .(.I1-! 114. ...“? I .4. I. . 0.. 0,. .04 I... . 0.. 00.1004. .. 4.1-0. 0 4. . . . . ‘I 4 .I.!I I I lot-004.0). -.0 .I I: 4. IL ..I. I 0 4 0 o. n . I 4 n L ..L: . I. 1.0 . .I I 07 0 .0 . .I.. 4'0\\4.0 .0. L ..0 0.900 .I..I40I 0.10. 3114...! .l-Io '50‘VLII‘ ,.._¢..... 3- .II ..I .... I00, I L. .I....I ... . . ... 1002‘. .I ....r... a p I... 4 .. . .1320...1 . . . I .. .. 0 . .. .0 1 .4. . . - . .I . -. .. . . . . .I. ..I -.00 I . I .4 . o. I. .0- .V . 1..... .. .4 01-033. .0. ... 3.70.0...II034- 451.35.450.00; .0f1.0.5 ...I..... .315. .I. .. on. .I., .. u 4 . ...4 I. .. . 10.. o . v. I: I. ..0 ..0. I .4. o. 54 .... I .....0. 00 .., . .. .. . .- x a. . ...: .. .. ... 4-. J... . . 0 I I : . 0. . V I I .I ... o ._ 44 40 0.. 4 0.40.03, 0.00. 0.... 0-0 ..0-14110.1 14140102. ..0 .... o I... .I- - I0 4 . 404-. .-... 44.10.01 . . II .0,-000- .. 4. 0,...04- II....! 4. 4 - -. II .II... o. . .. . U. :4..- 1 04. ... . .L .. I? . 4 4 I I. . I I no. 4. . 0 \I . 0 0. Ir: . 0.0 I . I I0. .11).. 0.14 1.4.10.4 40441-1,- 3.4)..0 1.11.“..04..«..‘.‘.0.L ..0‘ I ..0 .0. 1.1.... V ..II. 0.4... .4; O. ..,-..IIE 40.. 0 00. .4 .- . . ... 1!..0 .0 - . 00 .I.‘ 1.0 . I .01 ‘ I 4 94. I n 1 III. 4. Vs. I I .I.. 00. '- 1. .III .0617..001(-0.09014'1I91‘o. 4.10%) ....n. LI..4.:I 31..., .. . ...0: ...4 ...:I. . 1.3.41: 0..-4!I ... ... -. .0... ...... -1.. ., 1 .-....I 0 .. I0 .4 . ... .0 I... .- 4-.0 4 .4 .4 .II. 4.41. 1.0-...1 0 - 4...... I.-..-I.I.0\ 3-1031. 0. . 4‘ 44.5.0 ... -400!» ...0; ..0 {...-0040. ,4. ...... I ‘ .2... . .I. .- ... ... . . .\ .1k 4. L .I 7 . .. o v I . u . 0 .. l . I. _. I . - I. , . 10.1. 0. I.- . I o . .I. - '4 .41. - II . .0 .400 .10. o 0! ....l . I10 . 40-400. ‘10‘0-I‘0t‘1’IzJ‘ ..o, .-. . 0 .. 0.40 .01 0 o J. 4.........'...‘ . I ...0 I. . . n .4 .4. .l\ .... . ..... p .I .0. 4.: 4.4. ....0. . 0 . s . ..0-... . .....-I I-- . - I... . I . . . . - .44.... I 0.! 0|... .-4. I 0..-,. ... 0.. 0. . a .I. o. 4... .. «II. ...: vII.-0 o... I) A .- ... .41 .40 .../111.01.}...14170001410311.10‘401‘4'11110100 I00... ...I404043. >4 0 ......4 4. 1. .00..»0. .. 00‘. an: 44 0.1...- o . : 7.. 4 ...141..... .0 ..,. . J- 1; I .. I..- I o! I .0 .4. . . x. P . . ., 3 ... . . f. ... v.0.- .. .I. 0 . - . 4 4 .0 .4.... 0.4. 0.. . 4. C 14.. . II0.\ ...- LxI-QI-rlx“ 0\1101!. ..1.I-0I' 1107011; 0014:. II I. ..u I- . ..0. .0: III ..4 .".4I .. o 4. - -. 0. I10 - ...054+ 44 .. . .I . 00. o|01.1 uI supp-.... . I. .4. I ..0 I . 0- 4. ...II III I 1‘. .o o A . 0- 0 4. . 0 ~ I . .I. \. .. ..4 V I A . ..I 4 I 4 04‘00 .0- I «5.44..- .0... ., .I. 10".,1 1‘0. I-‘III- I “1%.. , _..II.._ ....0.. - .... 4.I ... .. f . .f .010... . . . o. 44 . - 4 4 . .. I I . I I 4 I. . -. ... .. ... . 0 . 0 -I .. . . ..1 «I ...I 0 4,140.14 .40 -I -. 4“. .I .100 .. | I - . i. ..1 I HM .- n ..I 4.. . .. 4. I r 1 < ... . . 4 . . .. 4 ...0 - . I .0 {I03 .11.. - . I. I1 .0 1 L I 0 I. .4 . I04I.. Ilkaio3,.,0'VIIIIH-‘I.I\.‘C,‘ILI.QCBIIJ\.10.I1|‘I-"fi0111120400 . . 04 1 . . 0. I 4 .I. I 4 - .4 I. I 0 0| . 4 n . . 3 I44 I o .0 . 4- 1.0.. I00- I I10. {.I.-,t‘cI-u ...0- .LII‘?IL..IL 0‘00 .0:I|\00‘01’...01) ‘0001.‘ . l . I I . II . .0 a... ...0 l 0 ..l. 1.4.0. \ .IIILI. {-9014-13-3 II. T "1- (.I..II..14I1I4.. 0-14.!" .I 1- I . I . .0 4% IVII V..I-h.’tl.. 0I1‘I-VII 3140.41.- 0.1.. I I4. I) 14 '10... I! ....xéfif9..rt-L. I4.II.. . -- fun-w fire - .- ..0 H. .... .I.-5|- ..fi..§fl.¥fl:1w3§ifl.§m§fi ..é ..m....r.m£~.g.¢.r5§.:-Kn.4 ......21... .91.»-..11-0-ufi ...flWnauno. www.myMwfiJ «flan... In? It- -4.:. I I: ...- 4I.......I I}... I_. ,.I....,-..\.. :34]. L . .I . . t.~.50-40..1:.... . . J4 ... .V I. Rho-:02... . .. . S 4. nun I4.....\.. I... I .1044. I ... 4 .I. ..., 4 .. ... ,.. 4 - .I .0 I.» . .... 0.- 8.. 0. . . 9.- 1 OJ”, LYLE-0 0:..0.I..- $4M ?m:fiw.i; .gw $A.3-WL1‘0W 0. I.‘ 1g.” “flmmwh wqu;w..I.-u§..1.mwwkn,ommumi cum-... “Ruin-mm.oofiufifltmgfm‘MJ-uutz 4'. 0.Wu..141.£w_4 .m. «A; ”III III I '- I.I III...) . . . ... III-III .0. 1..... 1941.0. LIBRARY Michigan State University This is to certify that the dissertation entitled CRYSTALLOGRAPHIC AND PROTEIN ENGINEERING STUDIES OF THE STRUCTURE AND FUNCTION OF CYTOCHROME C OXIDASE FROM RHODOBACTER SPHAEROIES presented by JIAN LIU has been accepted towards fulfillment of the requirements for the Ph. D. degree in CELL AND MOLECULAR BIOLOGY Wr Professor’s Signature DEC /-é , 29/ C3 1 Date MSU is an Affirmative Action/Equal Opportunity Employer PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K:/ProilAcc&Pres/CIRCIDaIeDue.indd CRYSTALLOGRAPHIC AND PROTEIN ENGINEERING STUDIES OF THE STRUCTURE AND FUNCTION OF CYTOCHROME C OXIDASE FROM RH ODOBA C T ER SPHAEROIDES By J ian Liu A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirement for the degree of DOCTOR OF PHILOSOPHY Cell and Molecular Biology 2010 ABSTRACT CRYSTALLOGRAPHIC AND PROTEIN ENGINEERING STUDIES OF THE ATESTRUCTURE AND FUNCTION OF CYTOCHROME C OXIDASE FROM RHODOBA C TER SPHA ER OIDES BY Jian Liu Cytochrome c oxidase (CcO) is the terminal enzyme of mitochondrial and bacterial respiratory chains. It accepts electrons from cytochrome c and reduces oxygen to water. Driven by this process, protons are translocated across the mitochondrial inner membrane of eukaryotes or the periplasmic membrane of prokaryotes, forming a transmembrane proton gradient that is used for the synthesis of ATP. Cytochrome c oxidase from Rhodobacter sphaeroides (RchO), a homologue of mammalian CcO, is used as the research model for structural and functional studies in this thesis. A RchO-EYFP (Enhance Yellow Fluorescent Protein) fusion protein was created to increase the hydrophilic portion of CcO and form better crystal contacts. Activity and proton pumping assays showed that the fusion protein was fully active. Reconstituted vesicle results also showed that the pH-sensitive EYFP in this fusion protein could be used as a pH indicator for the study of the proton pumping mechanism. Two mutants of RchO with different shortened subunit I C-termini were constructed and crystallized. The 16 residue deletion mutant (PJL33) showed lower activity at high pH and weak proton pumping, suggesting that the deletion affected the stability of the subunit III interaction with the enzyme. The crystal structure of PJL33 (2.10 A) also showed a conformational change at the new C-terminus, which could contribute to the activity changes. The 6 residue deletion mutant (PJ L49) showed normal activity except a diminished proton pumping function. The crystal of PJL49 diffracted to 2.5 A. In neither case did the removal of flexible regions result in higher resolution four subunit crystals, as hoped. High resolution crystal structures were obtained of the two mutants that define the proton uptake pathways in Rch0, D132A and K362M. In the oxidized crystal of D132A (2.15A), the mutation caused no change in overall structure, but a localized conformational change in the D132 region. A chloride ion replaced the D132 carboxyl position and the backbone of residues 130 to 135 shifted, causing a conformational change of N207 and loss of its bound water. These changes could block proton uptake in the D-pathway and account for the strong inhibition. The oxidized crystal structure of the proton path mutant, K362M (2.30 A), showed no significant overall structural changes, in spite of major inhibition, except for the loss of two waters adjacent to K362 and T359. This result supports the critical role for water molecules in proton uptake. Crystal structures of reduced forms of both K362M (2.50 A) and D132A (2.15 A) showed similar conformational changes as seen in WT, supporting the importance of redox-induced conformational effects. Spectra taken during X-ray radiation of the oxidized crystals showed reduction of the metal centers, but indicated a strained configuration that only relaxes to a native reduced form upon annealing. The results explain the ability to observe conformational differences between oxidized and reduced crystal forms. ACKNOWLEDGEMENTS First 1 would like to express my sincere thanks to my mentor, Dr. Shelagh Ferguson-Miller, for her kind support and guidance on my Ph. D. research during these years. Her wisdom, knowledge and enthusiasm in science have always inspired and motivated me. It is impossible to finish this dissertation without her. I am very grateful to my graduate committee members Dr. Robert Hausinger, Dr. David Weliky, Dr. Zachary Burton and Dr. Gregory Zeikus, for their advice and support throughout my Ph. D. program. I also want to thank my present and past lab mates for their valuable help and priceless friendship. I thank Dr. Carrie Hiser for all the guidance on the molecular biology techniques and for reading through my dissertation. Several important strains that were used in this dissertation were made by Dr. Carrie. I thank Dr. Denise Mills for the advices on making of the oxidase vesicles and she taught me how to use the stop-flow instrument. I thank Dr. Ling Qin for the guidance on crystallography. He taught me from the growing of crystals to the analysis and refinement of the crystal data sets. I thank Dr. Martyn Sharpe for the discussions on the mechanisms involved in cytochrome c oxidase. I also want to thank Dr. Bryan Schmidt, Dr. Namjoon Kim, Dr. Xi Zhang, Dr. Shujuan Xu, Fei Li and Leann Buhrow for their help and discussions. Dr. Namjoon Kim also helped me on the research of oxidase vesicles. Dr. Xi Zhang helped me to understand the mass spectrometry data of cytochrome c oxidase. I thank Fei Li and Leann for the advices and helping on defense presentation preparation. 1 also thank all the past and present iv undergraduate students who have helped me growing bacteria, making reagents, and preparing cell membranes. I also want to thank Dr R. Michael Garavito and Dr. Kaillathe Padmanabhan (Pappan) for the guidance and advices on the crystallography. Members of the Dr. Garavito Lab, Yi Zheng and Yanfeng Zhang also gave me lots of help during the synchrotron trips. Dr. Kaillathe Padmanabhan also helped me with computer hardware and software problems. I also thank Dr. Neil R. Bowlby and Dr. Steve A Seibold for the discussion and advices on the research of cytochrome c oxidase. I thank staff scientists at LS-CAT, GM/CA-CAT, and BioCars at Advanced Photon Source, Argonne National Laboratory, particularly Dr. Joseph S. Brunzelle and Dr. Zdzislaw Wawrzak for their training, Dr. Vukica Srajer and Dr. Yu-Sheng Chen for the support and helpful suggestions on online spectra data collections. Finally I would like to thank my parents and my sister, for their support and encouragement. Thank you all for everything. TABLE OF CONTENTS LIST OF TABLES ................................................................................ ix LIST OF FIGURESX LIST OF ABBREVIATIONS .................................................................. xiii CHAPTER 1. INTRODUCTION ................................................................... 1 1. BACKGROUND ON CYTOCHROME C OXIDASE ................................................................ 1 1.1 Energy metabolism and the respiratory chain ...................................................... I 1.2 Cytochrome c oxidase and heme-copper oxidase superfamily ............................. 3 1.3 Comparison of bovine heart CcO and bacterial CcO .......................................... 4 1.4 The structure of RchO ......................................................................................... 9 1.4.1 Overall structure of RchO ............................................................................ 9 1.4.2 Non-redox active metal centers ................................................................... 14 1.5 Electron sources and pathways .......................................................................... 15 1.6 Proton uptake channels: D- pathway and K- pathway ....................................... 18 1.6.1 The D proton pathway ................................................................................. 18 1.6.2 The K-pathway ............................................................................................. 20 1.6.3 The H proton pathway in bovine heart CcO ................................................ 21 1.7 Oxygen pathway in CcO ..................................................................................... 21 1.8 The water/proton exit pathway(s) ....................................................................... 22 1.9 Reaction cycle of RchO ..................................................................................... 23 1.10 Proton pumping and electron transfer coupling mechanism ............................ 25 1.11 The Regulation of CcO ...................................................................................... 27 2. MEMBRANE PROTEIN CRYSTALLOGRAPHY ................................................................ 28 2.1 Challenges in membrane protein crystallography .............................................. 29 2.2 Progress in membrane protein crystallization .................................................... 31 2.2.1 Crystallization of membrane proteins in lipid cubic phase .......................... 31 2.2.2 Antibody assisted membrane protein crystallization ................................... 32 2.2.3 Fusion protein strategy to assist membrane protein crystallization ............. 32 3. BACKGROUND OF GREEN FLUORESCENT PROTEIN ..................................................... 33 3.1 Structure of GFP ................................................................................................. 35 3.2 GFP derivatives .................................................................................................. 35 3.3 Usage of GFP and its derivatives ....................................................................... 3 7 CHAPTER 2. MATERIAL AND METHODS ............................................. 39 1. MOLECULAR ENGINEERING OF RHODOBACTER SPHAEROIDES ..................................... 39 2. CELL GROWTH ........................................................................................................... 39 3. PREPARATION OF R. SPHAEROIDES CYTOPLASMIC MEMBRANES ................................. 41 4. UV-VISIBLE SPECTROSCOPY ..................................................................................... 41 5. PROTEIN CONCENTRATION ASSAY ............................................................................. 42 6. SOLUBLIZATION OF THE MEMBRANES ........................................................................ 42 7. SDS-PAGE ................................................................................................................ 43 8. PURIFICATION OF THE RSCCO .................................................................................... 43 vi 8. 1 Ni-NT A purification of the Subunit 1 Histidine-Tagged RchO ........................ 44 8. 2 Ni-NT A Purification of the Subunit II Histidine-Tagged RchO ........................ 44 8. 3 Ion-exchange Column Purification of RchO ................................................... 45 9. RECONSTITUTION OF CYTOCHROME C OXIDASE INTO VESICLES ................................ 46 10. CYTOCHROME C OXIDASE ACTIVITY ASSAY ............................................................ 47 1 1. CRYSTALLIZATION OF THE RchO ........................................................................... 48 11.1 F our-subunit RchO Crystallization ................................................................ 48 11.2 T wo-subunit RchO Crystallization ................................................................. 50 12. FLASHCOOLING OF THE RSCCO CRYSTALS .............................................................. 51 13. DATA COLLECTION AND PROCESSING ...................................................................... 52 14. MOLECULAR REPLACEMENT AND STRUCTURAL REFINEMENT ................................. 52 CHAPTER 3. CONSTRUCTION OF A FUSION PROTEIN OF RSCCO WITH A GREEN FLUORESCENT PROTEIN VARIANT FOR CRYSTALLOGRAPHY AND PROTON PUMPING NIECHANISM STUDY .......................................................................................................... 54 1. INTRODUCTION .......................................................................................................... 54 2. METHODS AND RESULTS ............................................................................................ 55 2.1 Expression of GFP under the control of CcO subunit 1 promoter in Rs ............. 5 7 2.2 Expression of the Cc0-EYFPfusion protein in R. sphaeroides ......................... 60 2.3 Purification of CcO-E YFP fusion protein ........................................................... 64 2.4 Activity assay of RchO-E YFP fusion protein .................................................... 68 2. 5 Crystallization attempts and results .................................................................... 68 2.6 Use of GFP mutants as indicators of pH in reconstituted vesicle to study the proton pumping mechanism ...................................................................................... 68 2.6.1 Reconstitution of COVS with CcO-EYFP fusion protein ............................ 72 2.6.2 Activity assay and RCR of EYFP-COVS ..................................................... 72 2.6.3 Preliminary fluorescence stop-flow analysis of EYFP-COV ...................... 74 3. DISCUSSION ............................................................................................................... 77 4. SUMMARY AND CONCLUSIONS ................................................................................... 78 CHAPTER 4. STUDY OF THE SUBUNIT I C-TERMINU S MUT ANT FORMS ......................................................................................................... 80 1. INTRODUCTION .......................................................................................................... 80 2. METHODS AND RESULTS ............................................................................................ 84 2.1 Construction of the short subunit 1 CcO mutants PJL33 and PJL49 ................. 84 2.2 Protein expression and purification of PJL33 and PJL49 .................................. 84 2.3 SDS-PAGE of purified PJL33 and PJL49 Cc0 .................................................. 88 2. 4 UV-vis spectra of purified PJL33 and PJL49 CcO ............................................. 88 2.5 Activity of PJL33 and PJL49 .............................................................................. 88 2. 6 Proton pumping assay results of PJL3 3 ............................................................. 91 2. 7 Construction of subunit 1 C-terminus single mutant E552A ............................... 96 2.8 Expression and purification of E552A ................................................................ 96 2.9 Activity assay of E55 2A ...................................................................................... 96 . 2.10 Crystallization of PJL33 and PJL49 ............................................................... 102 vii 2. 11 Crystal structure of PJL33 ............................................................................. 104 2.12 Crystal structure of PJL49 CcO ..................................................................... 107 2.13 Construction and Preliminary stuay of the subunit [-1]! fusion protein .' PJL63 ................................................................................................................................. 1 0 7 3. DISCUSSION ............................................................................................................. 11 1 4. CONCLUSION AND SUMMARY .................................................................................. 114 CHAPTER 5. CRYSTAL STRUCTURES OF PROTON UPTAKE PATHWAY MUTANTS OF RSCCO ........................................................ l 16 1. INTRODUCTION ........................................................................................................ 1 16 2. METHODS AND RESULTS .......................................................................................... 1 19 2.1 Plasmid construction ........................................................................................ 119 2.2 Protein expression and purification .................................................................. 119 2.3 Crystallization of D1 32A and K3 62M .............................................................. I 19 2. 4 Crystal structure of oxidized D132A ................................................................. 123 2.4.] Overall structure of oxidized D132A ......................................................... 123 2.4.3 N207 conformation change and loss of water in D132A ........................... 127 2. 5 Structure of oxidized K3 62M ............................................................................ 128 2.5.1 Overall structure of oxidized K362M ........................................................ 128 2.5.2 Changes in K-pathway in K3 62M oxidized crystal ................................... 128 2. 6 Reduction of D1 32A and K3 62M crystals ........................................................ 130 2. 7 Structure of reduced crystals of D1 32A and K3 62M ........................................ 130 2.8 On-line microspectrophotometric analysis of single RchO crystals .............. 134 3. DISCUSSION ............................................................................................................. 137 4. SUMMARY AND CONCLUSIONS ................................................................................. 138 APPENDIX ................................................................................................. 1 4O BIBLIOGRAPHY ....................................................................................... 1 43 viii LIST OF TABLES Table 2. 1: Mutant strains created and used in this thesis ................................................. 40 Table 3. 1: Activity of FPLC purified RchO-EYFP fusion protein ................................ 69 Table 3. 2: RCR measurement results of RchO-EYF P COV ......................................... 73 Table 4. 1: Activity of PJL45 CcOat pH 6.5 and pH 8.6 ................................................ 100 Table 4. 2: Crystal parameters of F] L33 and PJ L45 ....................................................... 103 Table 5. 1: Crystallization conditions of D132A and K362M ........................................ 121 Table 5. 2: Crystal parameters of D132A and K362M ................................................... 122 ix Figure 1. Figure 1. Figure 1. Figure 1. Figure 1. Figure 1. Figure 1. Figure 1. Figure 1. Figure 2. Figure 3. Figure 3. Figure 3. Figure 3. Figure 3. Figure 3. Figure 3. Figure 3. Figure 3. Figure 3. Figure 3. LIST OF FIGURES 1: The mitochondrial respiratory chain (Electron Transfer Chain). .................... 2 2: Comparision of bovine heart CcO structure and bacterial CcO structure. ...... 7 3: Conserved lipid binding between bovine heart and bacterial CcO. ................ 8 4: Overall structure of RchO. .......................................................................... 10 5: Homes and metal centers in Rch0 and their amino acid ligands. ............... 13 6: Proton uptake pathways in Rch0. ............................................................... 19 7: Reaction cycle of Rch0 The green circle is the CcO. ................................. 24 8: Structure of Green Fluorescent Protein. ........................................................ 34 9: A proposed mechanism of the formation of the chromosphere of GFP ........ 36 1 Crystallization setup for the RchO ................................................. 49 1: GFP pH sensitive mutants. ............................................................................ 56 2: Plasmid construction of pJL-4 ....................................................................... 58 3: Expression of EYFP in R sphaeroides. ......................................................... 59 4: Plasmid construction oprL-16 ..................................................................... 61 5: Expression of Subunit I-EYFP CcO fusion protein. ..................................... 62 6: Reduced spectra of FPLC purified RchO-EYFP fusion protein. ................ 63 7: Fluorescence spectrum of EYFP and CcO-EYFP fusion protein .................. 65 8: Proteolysis of CcO-EYFP protein ................................................................. 66 9: Reconstituted vesicles of RchO-EYFP ........................................................ 7O 10: Cytochrome c oxidation by WT COVS and CcO-EYFP COVS. ................. 75 11: Proton pumping by RchO-EYFP COVS .................................................... 76 Figure 4. 1: C-terminus comparision of 4-subunit RchO crystal and 2-subunit CcO crystal. ....................................................................................................................... 81 Figure 4. 2: Possible interaction between subunit I and subunit III of RchO. ................ 83 Figure 4. 3: Construction of pJL-33 expression plasmid .................................................. 85 Figure 4. 4: Construction of pJL-49 expression plasmid .................................................. 86 Figure 4. 5: Reduced -minus-oxidized Spectrum of the membrane fraction of PJL33 and PJL49 CcO ................................................................................................................ 87 Figure 4. 6: SDS-PAGE of FPLC purified PJ L33 and PJL49 protein .............................. 89 Figure 4.7: Spectrum of FPLC purified PJL33 and PJL49 CcO ....................................... 90 Figure 4. 8: Activity curve of PJL33 and PJL49 compared to WT CcO .......................... 92 Figure 4. 9: Suicide inactivation of PJL33 CcO ............................................................... 93 Figure 4. 10: Cytochrome 0 oxidation of PJL33 measured by stopflow. ......................... 94 Figure 4. 11 Proton pumping assay results of PJL33 COV and WT COV ....................... 95 Figure 4. 12: Primers used in the construct of PJL34: E552A mutant. .......................... 97 Figure 4. 13: Reduced minus oxidized spectra of PJL45 membrane fraction. ................. 98 Figure 4. 14: Spectra of purified PJL45 CcO. .................................................................. 99 Figure 4. 15: Proton pumping assay of PJL45 COV. ..................................................... 101 Figure 4. 16 Structure alignment of the redox centers of PJL33 with WT CcO ............ 105 Figure 4. 17: Structure comparsion of PJL33 and WT CcO at C-terminus region ......... 106 Figure 4. 18: Structral comparison of the C-terminus region of WT COD and PJL49 108 Figure 4. 19: Reduced-minus-oxidized spectra of P1 L63 membrane fraction ................ 109 Figure 4. 20: Reduced and oxidized spectra of FPLC purified PJ L63 ........................... 110 xi Figure 5. Figure 5. Figure 5. Figure 5. Figure 5. Figure 5. Figure 5. Figure 5. Figure 5. : Proton uptake pathways in Rch0 .............................................................. 117 : Spectra of membrane fraction of D132A and K362M. ............................... 120 : Redox centers in oxidized D132A crystals. ................................................ 124 : Comparison of D-pathway water arrangement in D132A and WT CcO 125 : The mutation region in D132A oxidized crystals ....................................... 126 : Structural changes in K362M. ..................................................................... 129 : Heme a3 region of the reduced crystals of RchO ..................................... 131 : Alternative conformations in D132A .......................................................... 133 : Online spectra of single crystal of RchO ................................................... 135 xii ATP A. U. BCA BSA CcO CCCP COV DEAE DDM DM EDTA EYFP FADHz FCCP F PLC FRET F ITC GFP HEPES LDAO ABBREVIATIONS adenosine triphosphate asymmetric unit bicinchroninic acid bovine serum albumin cytochrome c oxidase Carbonyl cyanide m—chlorophenyl hydrazone cytochrome c oxidase vesicle diethylaminoethyl dodecyl maltoside decyl maltoside ethylenediaminetetraacetic acid enhanced yellow fluorescence protein reduced form of flavin adenine dinucleotie trifluorocarbonylcyanide phenylhydrazone fast protein liquid chromatography FOrster /fluorescence resonance energy transfer fluorescein isothiocyanate green fluorescent protein 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid kilodalton lauryl dimethylamine n-oxide xiii MD MES NADH Ni-NTA PCR Pd PEG Rs SDS-PAGE Tris RCR WT molecular dynamics 2-(n-morpholino)ethanesulfonic acid reduced form of nicotinamide adenine dinucleotide nickel-nitrilotriacetic acid Nuclear magnetic resonance polymerase chain reaction Paracoccus denitrificans polyethylene glycol Rhodobacter sphaeroides sodium dodecyl sulfate polyacrylamide gel eletrophoresis tris (hydroxymethyl) aminomethane respiratory control ratio wild type xiv CHAPTER 1. INTRODUCTION 1. Background on cytochrome c oxidase 1.1 Energy metabolism and the respiratory chain Energy metabolism is a very important aspect of all living organisms. From simple organisms such as bacteria to complicated eukaryotes such as mammals, a continuous supply of energy is always required to perform all the necessary functions to support life. Through catabolic metabolism, energy-containing nutrient molecules such as carbohydrates, fats, and proteins are degraded into smaller, energy-depleted products such as H20, C07,, and NH3. The energy released in this process is then stored in the form of ATP and reduced electron carriers (N ADH, NADPH, and F ADHz). Catabolism consists of sequential metabolic pathways including glycolysis and fatty acid oxidation, citric acid cycle, and oxidative phophorylation to produce a useful chemical energy currency, ATP. Oxidative phosphorylation is a highly efficient energy-converting process. It involves the reduction of 02 to H20 with electrons donated by NADH and FADHz. The electron flow takes place through the respiratory chain (electron transfer chain) in the inner membrane of mitochondria in eukaryotes, or the cytoplasmic membrane in prokaryotes. The free energy generated by electron flow in the respiratory chain is conserved in the form of a pH gradient and transmembrane electrochemical potential. The respiratory chain consists of a series of transmembrane protein complexes (Shown in Figure 1.1). Complex I (N ADH dehydrogenase) and Complex II (succinate dehydrogenase) catalyze electron transfer to ubiquinone from soluble electron donors such as NADH and Cytochrome c NADH+ NAD ' Complex I Complex II Complex Ill Complex IV F1 Fo-ATPase Cytochrome c Oxidase Figure l. 1: The mitochondrial respiratory chain (Electron Transfer Chain). The respiratory complexes are located in the innermembrane of mitochondria. Electrons from food sourcesare transferred to Complex 1 (NADH dehydrogenase) or Complex II (succinate dehydrogenase) through NADH and FADHz, respectively. Then through the coenzyme Q transfers the electrons to complex HI (bcl complex). Cytochrome c then transfers the electrons from complex III to complex IV (000), which was used to reduce dioxygen to form water. Complexes 1, III and IV are known to translocate protons across the inner mitochondrial membrane. The proton gradient formed across the inner mitochondrial membrane is then utilized by Complex V (ATP synthase) to generate ATP. (Images in this dissertation are presented in color). succinate (Yagi et a1. 1998; Albracht and Hedderich 2000; Iverson et a1. 2000). Complex 111 (cytochrome bc; complex) transfers electrons from ubiquinol to cytochrome c (Berry et a1. 2000; Darrouzet et al. 2000). Complex IV (cytochrome c oxidase, EC 1. 3. 9. 1) ends the sequence by transferring electrons from reduced cytochrome c to 02 (Ferguson-Miller and Babcock 1996). Complexes 1, III, and IV couple electron transfer to proton translocation across the membrane from the mitochondrial matrix or bacterial cytoplasm to the interrnembrane space. For each pair of electrons transferred through the electron transfer chain to 02, four protons are pumped out by Complex 1, four protons by Complex 111, and two protons by Complex IV. The energy stored in such a gradient is called the proton-motive force and consists of two different potential energies: the chemical potential energy due to the different concentration of protons separated by the membrane and the electrical potential energy due to the separation of charge when a proton moves across the membrane without a counter ion. This electrochemical energy is finally used by the ATP synthase to produce the energy currency of living organisms, ATP. 1.2 Cytochrome c oxidase and heme-copper oxidase superfamily As Shown in Figure 1.1, cytochrome c oxidase (CcO) is the terminal enzyme in the respiratory chain. The function of C00 is to catalyze the reduction of oxygen to water and pump an additional proton across the membrane for each electron and proton consumed in the reaction. The overall reaction can be expressed as: 4 cyt c2+ + 8H+(in) + 02 -> 4 cyt c3+ + 2H20 + 4H+(out) In this way, a transmembrane electrochemical gradient is produced (F erguson-Miller and Babcock 1996; Michel et a1. 1998). CcO belongs to the heme/copper oxidase superfamily. This superfarnily also includes many bacterial heme/COpper oxidases, which are defined by the presence of a catalytic core containing the oxygen binding heme a3 and CuB (Garcia-Horsman et a1. 1994). The variation in heme/copper oxidases is due to the differences in the active site and proton channels. A- and B-families (Chang et a1. 2009) need either an 0 or a heme in the active site, both of which have a famesyl tail, whereas the C-family utilizes b heme (with no tail) in the active Site (Sharma et al. 2008). The catalytic substrate can either be cytochrome c (in the case of CcO) or quinol, as in the case of cytochrome b03 quinol oxidase from E. coli. In Rhodobacter sphaeroides (Rs), there are three types of CcOs: the M3 type, the cbb3 type and the mag, type. The most similar form to the mitochondrial CcO in Rch0 is the am type. In this thesis, the research is all based on the am from RchO. 1.3 Comparison of bovine heart CcO and bacterial CcO The position of CcO as the terminal member in the respiratory chain defines the importance of this enzyme, which consumes more than 90% of the oxygen we breathe. Historically, bovine heart CcO has been the best studied research model, due to the cow heart being both large and a very rich source of mammalian Cc0. The X-ray crystal structure of bovine heart Cc0 shows that it can be a dimer in the purified state (Tsukihara et a1. 1996). It is a 13-subunit transmembrane complex. The catalytic core subunits (subunit I, II and III) are encoded by the mitochondrial DNA and ten others subunits are encoded by the nuclear genome (Kadenbach et al. 1987). Extensive crystallographic studies have been carried out on the bovine heart CcO, including many different forms such as the fully oxidized, fully reduced, azide-bound, cyanide-bound and carbon monoxide—bound (Yoshikawa et al. 1998). Some structural changes have been observed in the crystal structures of bovine heart CcO, revealing possibly important mechanisms for proton uptake. For example, the Asp-51 in subunit I was seen to undergo a conformational change in reduced compared to oxidized crystals. This finding led to the proposal of an additional proton channel for pumping in the mammalian CcO, the H-channel, which is not conserved in bacteria. A considerable amount of internal water is also observed in the bovine heart CcO, thanks to the high resolution (1.8 A) crystal structure (Tsukihara et a1. 2003). These waters could be involved in the proton pumping mechanism, especially in the D-pathway. The X-ray structure of bovine heart Cc0 also reveals the important role of lipids. In the crystal structure, thirteen lipids, including two cardiolipins, one phosphatidylcholine, three phosphatidylethanolamines, four phosphatidylglycerols and three triglycerides, are identified (Shinzawa-Itoh et a1. 2007). These lipids may play a critical role‘not only in the structural stabilization of the dimer and the whole enzyme, but also in 02 transfer to the active site and other as yet undetermined fimctions. Although the study on bovine heart CcO can provide much information and insights on Cc0, it is extremely difficult to handle when it comes to Site-directed mutagenesis for functional and mechanistic analysis, due to the fact that the largest catalytic subunits are encoded in the mitochondrial genome, which has different codons for a number of common amino acids. The bacterial CcO, such as RchO, is a good model system for mechanisic studies because the catalytic core subunits (I and II) are highly homologous to their mammalian counterparts (Figure 1.2) and ease of Site-directed mutagenesis and expression in the bacterial system allows the production of numerous mutants to test functional hypotheses. In R. sphaeroides, its ability to obtain energy from photosynthesis, as well as possessing multiple types of CcOs besides the M3 type, allows the production of Cc0 knock out strains with a clean background for the mutant Cc0 expression. The bacterial aa3-type CcOs are highly conserved, such as the Paracoccus denitrificans and R. sphaeroides CcOs (sequence similarity: 81% in subunit 1, 49% in subunit II, 69% in subunit III, and 48% in subunit IV), and are closely related to the bovine Cc0 (sequence similarity of Rs to bovine: 76% in subunit 1, 63% in subunit II, 71% in subunit III). The crystal structure of RchO shows as many as 178 water molecules forming hydrogen-bonded networks inside the enzyme and a high degree of conservation of ordered, hydrogen-bonded water networks in proton pathways, suggesting a critical role of water in proton transfer (Sharpe et a1. 2005). The RchO crystal structure also shows a total of six phosphatidylethanolamines in the four subunit Cc0 crystal structure (Svensson-Ek et a1. 2002), with four positioned on the interface between subunit IV and VIII, and two associated with subunit III and subunit 1. The two subunit crystals of Rch0 at 2.0 A resolution, although missing subunits III and IV and the six phospholipids associated with them, revealed the positions of many more lipid /detergent sites that also appear highly conserved in position compared with the bovine and bacterial homologs, including amino acid residues in their vicinity. These findings and those in other crystal structures suggest a specific role of lipid in membrane proteins (Qin et a1. 2006) (Figure 1.3). Bovine heart Cc0 Bacterial C60 Figure 1. 2: Comparision of bovine heart CcO structure and bacterial CcO structure. CcO from bovine heart contains 13 subunits (left) and the Cc0 from bacteria (Rs) contains 4 subunits (right). The dotted lines represent the lipid bilayer membranes. The catalytic core of the bacterial Cc0, subunit I (green) and subunit II (cyan), is highly homologous to that of the bovine heart C60. The subunit III (purple) is also a conserved subunit. 81106101396th (35} C60 RhodobacterCcO Rh odobactei; Paracoccus, W1th Upld/ detergent Overlayed on Paracoccus (Pd) Bovine (bov) CcO Overlayed Figure l. 3: Conserved lipid binding between bovine heart CcO and bacterial CcO. The lipids/detergent binding sites found in the 2 subunit crystal structures of bacterial CcO fi'om different sources (Rs, left; Pd, middle) are conserved. Comparison of the bacterial Cc0 structure with the bovine heart Cc0 also shows that these lipid binding Sites are highly conserved in position (right).The lipids/detergents fi'om Rch0 are shown in green (left) and deep blue color (middle and right). The lipids/detergents from PdCcO are Shown in cyan color (middle and right). The lipid/detergents from bovine heart CcO are shown in yellow (right). These results indicate the important role of lipids in the structure and function of CcO. 1.4 The structure of RchO 1.4.1 Overall structure of RchO RchO contains 4 subunits as shown in Figure 1. 4. Subunit 1 (blue) and subunit II (purple) form the catalytic core of the enzyme, performing both electron transfer and proton uptake/pumping functions. Subunit I is the largest subunit with a molecular weight of 62 kDa. It consists of 12 transmembrane helices roughly forming three arcs of 4-helices each, noted first in the original structure of the Paracoccus CcO (Iwata et al., 1995). This subunit contains the redox active centers, heme a and CuB-heme a3. It also contains two proposed proton uptake pathways: the D pathway and K pathway. Figure 1.5 shows the metal centers in subunits I and II. The iron centers of the two heme groups, heme a and heme a3, are buried inside subunit I, 15 A from the periplasmic side of the membrane. The angle between the two porphyrin rings of the heme a and heme a3 planes is approximately 104° and this angle is quite conserved among the M3 oxidases (Sharpe et a1. 2005) although its functional significance is not clear. The hydroxyethylfamesyl tail of heme a extends downward and remains in the hydrophobic core between the transmembrane helices while the hydroxyethylfamesyl tail of heme a3 bends sideways and penetrates to the lipid bilayer. Heme a has a low spin iron with two conserved axial histidine ligands, Hile2 and His421 in subunit I. Heme a3 has a high spin iron that is five-coordinated, with only one conserved axial histidine, His419, which is ligated to the iron on the opposite side (distal side) of the heme a; to the CUB center. The distance between the CuB ion and the heme a3 iron is approximately 4.9 — 5.1 A in the Subunit II Heme ala3 Subunit IV Subunit | Figure 1. 4: Overall structure of Rch0. The subunit I (shown in green) contains the CuB and heme a and heme a3 (red stick model). Subunit H (cyan) contains the CuA. Subunit ID is shown in cyan color. Subunit IV is the single helix structure shown in orange color. Between subunit III and subunit IV there are several lipid moles (shown in blue sphere and stick models). 10 structures of Cc0 from different Species (Harrenga and Michel 1999; Svensson-Ek et a1. 2002; Tsukihara et a1. 2003). In between CuB and heme a3 is the Site where oxygen binds and is reduced to water. CuB has three ligands, H13284, His333, and H334 in subunit 1. In high resolution crystal structures, H284 is found to form an unusual covalent bond to Y288 between the N82 and C82 atoms from the two residues, respectively (Ostermeier et a1. 1997; Yoshikawa et a1. 1998; Buse et a1. 1999; Tomson et a1. 2002). It has been suggested that this unique linkage between His-Tyr lowers the pKa of the tyrosine phenol group below that of free TerH allowing the deprotonated tyrosine radical even at physiological pH. This makes the tyrosine a potential electron and proton donor with the possibility of becoming a neutral radical during catalysis (Ostermeier et a1. 1997; Proshlyakov et a1. 2000). The density between the heme a3 iron and CuB in the oxidized form of the enzyme has been variously interpreted as a peroxide (Yoshikawa et al. 1998), or one hydroxide bound to the CUB and a water ligated to the Fe ion of heme a3 (Ostermeier et a1. 1997). The molecular weight of subunit II of RchO is around 32.9 kDa. The N-terminus of subunit II is composed of two transmembrane helices which forms a loop structure while the C-terminus forms an extra-membrane globular domain (Svensson-Ek et al. 2002) which is composed of a ten stranded B—barrel structure. This domain is involved in the binding of cytochrome c. Two mixed valance Cu ions form the Cup, center inside subunit II, which is the initial electron acceptor from soluble cytochrome c (Zhen et a1. 1999). The ligands for the Cu include Cy5252, Cys256, His260, Hi3217, and Met263 in subunit II as Shown in Figure 1.5. The two cysteines bridge the two copper atoms from two sides and the two sulfur atoms lie in the same plane as the copper atoms. The importance of this unusual copper center in the 11 functioning of the protein is not clear; a role in proton pumping has been proposed (Chan and Li 1990), but argued against on the basis of retention of proton pumping in mutants where the structure of the Cu center is significantly disrupted (Zhen et a1. 2002). In RchO, the polypeptide chain of subunit II is known to undergo proteolysis after translation. After the cleavage, the signal peptide (25 residues) on the N-terminus of subunit II is removed (Steinrucke et a1. 1987). Besides the N-terminal processing, there is also an‘incomplete proteolysis of the C-terminal 13 residues (Hiser et a1. 2001). The reason for the incomplete C-terminal proteolytic cleavage is still unknown. However, it seems that this cleavage does not affect activity and function of the enzyme (Hosler et a1. 1992; Zhen et a1. 1998) but does interfere with crystallization due to molecular inhomogeneity. Subunit III is also around 30 kDa (30.1 kDa). It is composed of seven transmembrane helices and forms a V-shaped cleft between two bundles of helices which appears to be designed to hold four lipid molecules that also interface with subunit 1, though only 2 are resolved in the four-subunit structure (Svensson-Ek et a1. 2002). The function of subunit III is still unclear. It may play a role in the structural stability of CcO. Research has Shown that loss of subunit III causes the suicide inactivation of Cc0 (Bratton et a1. 1999). Subunit IV is the smallest subunit (6.3 kDa) with a single transmembrane helix. The function of this subunit is unknown. Deletion of subunit IV of RchO does not affect the activity in any way that we can detect. The X-ray crystal structure studies, showing four lipids bound to it, indicate that subunit IV is important in lipid binding (Svensson-Ek et al. 2002). In the 2-subunit crystal structure, subunit III and subunit IV are lost during crystallization. However, the structures of subunit I and II are highly similar to those of the 4-Subunit crystal structure. 12 Subunit II Mg / fl) I H411 Subunit I Figure 1. 5: Hemes and metal centers in Rch0 and their amino acid ligands. The heme groups are colored in gray and the amino acid ligands are colored by the atom types (C: green; N: blue ; 0: red; S: yellow). Iron, copper, magnesium, and calcium atoms are colored in red, purple, blue and bluish green, respectively. The membrane surface of the periplasmic membrane of Rhodobacter sphaeroide is Shown as orange dotted line and the interface between subunits I and II is represented by a light blue curve. 13 1.4.2 Non-redox active metal centers A non-redox active Mg2+ ion is found in between subunit I and subunit II of RchO, lying at the end of a proposed water channel, approximately 12 A from the surface and immediately above heme a3. The liganding residues of this magnesium ion are well conserved, including Asp412, His411 and a Shared ligand with CuA, Glu254 of subunit 11. Three water molecules are also found binding to this magnesium. Its location suggests a role in stabilizing the interface between subunit I and subunit II, or a role in the water exit pathway. The natural Mg2+ site can be substituted with Mn2+ for EPR studies by growing the bacteria in high manganese. Using D20 added externally, and stopped-flow freeze-quench EPR measurements, complete proton/deuterium exchange at the Mn2+ site was observed in less than 11.4 ms at room temperature, suggesting an even more rapid equilibrium between the Mn Site and the bulk solvent (Schmidt et a1. 2003). The results suggest that the Mn2+/Mg2+ site is involved in proton and/or water movement during turnover. Because of the complex hydrogen-bonding network of waters in the vicinity Of Mn”, it is unclear where exactly water or protons are moving. The binding of extrinsically added metals, such as Zn2+ and Cd”, to Cc0 is interesting because they inhibit proton movement in many proton-dependent systems (Chemy and DeCoursey 1999). In Cc0, proton uptake is strongly inhibited by micromolar concentrations of Zn2+ or Cd2+ (Aagaard et a1. 2002; Mills et a1. 2002). The inhibition of CcO activity by Zn2+ or Cd2+, both in the purified detergent-solubilized form and in reconstituted vesicles, suggests both internal and external binding sites (Hosler et a1. 2006). A cadmium binding site was clearly identified at the entrance of the K-pathway, involving side chain atoms of E101 and H96 in subunit II, in two subunit isotropic crystals (2.0 A) of 14 Rs CcO (Qin et a1. 2006). This position at the bottom of subunit II had been previously suggested as the entry point for the K-pathway (Branden et a1. 2002). Other structures of the bovine CcO (Shinzawa-Itoh et a1. 2007) show Zn2+ binding at the entrance of the D-pathway, as also predicted from kinetic analyses. However, SO far there has not been a Site defined on the outside surface of CcO to account for inhibition by Zn2+ of the exit/backflow pathway. There is also a Cay/Na+ site found in the RchO. However, the function of this site is still unknown for Rch0. In bovine heart Cc0, the binding of the Ca2+to the Site that appears normally occupied with Na+ causes a heme a red shift (Nicholls 1975; Wikstrom and Saari 1975). 1.5 Electron sources and pathways The electron transfer pathway in Rch0 is quite clear now. The electrons are first transferred from cytochrome c to the CuA center, and then to heme a, finally to the binuclear center, CuB and heme a3, where 02 binds and is reduced to water (Ferguson-Miller and Babcock 1996; Michel et a1. 1998). In R. sphaeroides, there are two types of cytochrome c that have been considered as the substrates for Cc0: cytochrome C2, which is a soluble, mobile molecule, and cytochrome cy, which is a membrane-anchored protein. Research results Show that the latter one is the more likely physiological electron donor for the aa3-type Cc0 in R. sphaeroides (Daldal et a1. 2001; Daldal et a1. 2003). However, for experimental purposes the soluble horse heart cytochrome 0 serves as an excellent substrate for Rch0. From molecular modeling using horse heart cytochrome c, the binding site for cytochrome c is proposed to be on the outside 15 of the membrane on a concave surface created by the globular domain of subunit II and the adjacent flat surface of subunit I (Roberts and Pique 1999; Flock and Helms 2002; Maneg et a1. 2004). Protein docking studies (Roberts and Pique 1999) and chemical modification studies (Ferguson-Miller et a1. 1978) along with intensive site-directed mutagenesis analysis (Ferguson-Miller et a1. 1978; Roberts and Pique 1999; Wang et a1. 1999; Zhen et al. 1999) Showed that the binding of cytochrome c to the oxidase is mainly through electrostatic interactions between the two docking faces. Five lysine residues on cytochrome c, Lys8, Lysl3, Lys72 and Lys86/87 (horse heart cytochrome c numbering), form an interaction with the Glu148, Glu157, Asp195 and Asp214 residues of subunit II of C00 (F erguson-Miller et a1. 1978; Roberts and Pique 1999; Wang et a1. 1999; Zhen et a1. 1999). Research results also Show that a conserved Trp143 on subunit II could be the immediate electron acceptor from reduced cytochrome c (Witt et a1. 1998; Roberts and Pique 1999; Wang et a1. 1999; Zhen et a1. 1999). Mutation of W143 decreased the electron transfer rate to Cu by close to lOOO-fold without affecting the binding of cytochrome c to C00 (Wang et al. 1999; Zhen et a1. 1999). The Cult Site, composed of two mixed-valence copper ions, is located in the extramembrane domain of subunit II. The electron transfer rate from Cu to heme a is very fast (2.3 x 104 3" in bovine heart mitochondrial CcO (Pan et a1. 1993) and 9 x 104 s'1 in Rch0 (Wang et a1. 1999)). Two major electron transfer (tunneling) pathways between the Cu site and heme a have been proposed. One of the pathways starts at the ligand (Cys252). Along this pathway, 26 covalent bonds and 2 hydrogen bonds and one through space jumping are involved. However, results showed that for this pathway the electron transfer rate would be much Slower than the measured rate. 16 The other pathway starts with a hydrogen bond between the Cult ligand His260 in subunit II, which bonds to a carbonyl group of the peptide bond between two highly conserved arginine residues Arg481 and Arg482 in subunit 1, and then through Arg482 to one of the propionate groups in heme a. This pathway is composed of 14 covalent bonds and two hydrogen bonds (Iwata et al. 1995; Ramirez et a1. 1995; Regan et a1. 1998; Tan et a1. 2004). The importance of the residues along the pathway is supported by the site-directed mutants H260N and R482P, which showed an approximately 2000 fold Slower electron transfer rate between CuA and heme a (Wang et a1. 2002; Zhen et a1. 2002; Qian et a1. 2004). These results can be used to support the concept of a “through bond” pathway of electron transfer. Although the distance between Cut and the heme a3 is only about 1.5 A longer than that between Cu and heme a, evidence for significant rates of electron transfer between Cu and the heme a3-CuB binuclear center has not been found (Regan et a1. 1998). There are two possible electron transport pathways between heme a and heme a3. The electrons could either be transported through the closely approaching heme edges, or the histidine ligands to each heme connected through helix X (Regan et a1. 1998). The direct electron transfer between the two heme porphyrin rings would occur through “edge to edge” transfer Since the closest edge to edge distance between the two heme groups is only approximately 4.6 — 5.2 A in CcO from different organisms (Ostermeier et a1. 1997; Harrenga and Michel 1999; Svensson—Ek et a1. 2002; Tsukihara et a1. 2003; Tan et a1. 2004). The center-to-center transfer via the histidine ligands involves some through space jumps and a distance of approximately 13.5 A between the two heme Fe atoms. 17 1.6 Proton uptake channels: D- pathway and K- pathway In each catalytic cycle, four protons are taken up from the inner side of the membrane to react 02 and produce water; four other protons are taken up from the inside to be pumped to the outside of the membrane against the membrane potential and pH gradient (electrochemical gradient)(Ferguson-Miller and Babcock 1996; Michel 1998; Schultz and Chan 2001; Mills and Ferguson-Miller 2003). Two possible proton uptake pathways, D and K pathways have been proposed in Rch0 as shown in Figure 1.8 (Iwata et a1. 1995; Wikstrom et a1. 2000; Svensson-Ek et a1. 2002) . 1.6.1 The D proton pathway The D pathway was named after a conserved aspartate residue Aspl 32, which is located on the inner surface of subunit 1 and is considered to be the entrance of the D pathway because of its dramatic inhibition of activity when mutated and its location close to the start of a hydrogen bonded chain of waters leading toward the active site. The chain of waters is organized by a series of hydrophilic residues including N121, N139, N207, 8142, Y33, S201, S200, and ending at E286 near the CuB-heme a3 binuclear center. Changes to these residues have marked effects on enzyme activity and the proton pumping, in some case inhibiting and in others uncoupling the proton pump (that is, allowing activity without proton pumping). In the crystal structure of Rch0, the chain of water molecules is very well defined in this pathway (Svensson-Ek et a1. 2002; Qin et a1. 2006). The importance of the ordered water in the fimction and kinetics of this pathway are still being studied. Computational and experimental results suggest that the side chain of E286, at the interior end of the pathway close to the hemes, might be a regulatory “switch control,” undergoing a conformational 18 Mg! * R481 Sc 61 E *4 Hemea g “tug: , .4 ‘~;P-.,_ )qlk‘, E286 ,1‘ J“! Heme a3 ..,: r T v 3201 Y2“ Tess ... v :5:- I...;..K362 j '- N139 v...- $299 I ........... .E101" N121 , , Cd 3. r . D132 ‘ Figure l. 6: Proton uptake pathways in Rch0. The subunit I is shown in yellow color and the subunit Ill is in blue color. Red dotted line and the blue dotted line are the D-pathway and K-pathway respectively. The water molecules in the structure are shown in red spheres. The green dotted line is a proposed water/proton exit pathway. 19 change that directs a D-path proton to either the active site CUB-heme a3 or to the exit pathway to be pumped out (Konstantinov et al. 1997; Hofacker and Schulten 1998);(Sharpe et al. 2005). The proton transfer pathway after E286 is still unclear. However, a reduced crystal structure of RchO shows the disappearance of one water, W301, between E286 and heme a3 upon reduction that could break the connection of the D-path to the active site. At the same time, reduction leads to the rearrangement of the water molecules below the CUB-heme a 3 Site, suggesting increased water access from the K path and thus a possible alternating gating mechanism (Qin et al. 2009) . 1.6.2 The K—pathway. The K pathway was named after a conserved lysine, K362 of subunit 1, which has a profound effect on activity (<0.02% WT) and was predicted (and found) to be in the middle of a conserved transmembrane region. This region does not show an organized water chain, but does lead to the active site, and MD calculations predict that water can organize in it. The K-pathway in Rch0 appears to start with a glutamate residue E101 in subunit II, and continues through 8299, K362 and T359 in subunit 1, up to the binuclear center, where molecular oxygen is reduced to water (Braenden et al. 2002; Tomson et al. 2003) . This pathway ends with a tight (2.6 A) hydrogen bond between the hydroxyl group of Y288 and the carboxyl group from the farnesyl tail of heme a3. There is also a water associated with this bond and with T359 at the top of this channel. The K pathway is believed to transport substrate protons to the active site. Mutation of the K362 to methionine shows extremely low activity. Mutations of 8299 and T359 strongly inhibit the activity as well. Unlike the D132A CcO, whose activity can be stimulated by the presence of a transmembrane potential, the K3 62M mutant does not Show an increase of the activity with 20 the membrane potential (Fetter et al. 1995; Mills et al. 2000). This lack of enhancement of activity in the K path mutants suggests that the K pathway is not connected to the external surface via any reversible proton path and is exclusively used for substrate proton uptake. Yet it remains unclear which and how many of the four required substrate protons are supplied by each pathway, and when and how these protons are used in the oxygen reduction reaction, a central issue in understanding the coupling of proton pumping and electron transfer. 1.6.3 The H proton pathway in bovine heart CcO In bovine heart Cc0, an H pathway is proposed in addition to the D and K pathways, based on the high-resolution crystal structure (Tsukihara et al. 1996; Yoshikawa et al. 1998). This pathway connects the mitochondrial matrix side of the membrane to the outside via water pools and residues in close proximity to heme a. It is postulated to be an exclusive pathway for the pumped protons in mammalian CcO, due to there being little or no conservation of the residues involved in the bacterial enzymes. Indeed, mutagenesis results Show that some of the essential residues along this pathway had no effect on the bacterial CcOs, indicating that the H pathway is not likely to be a universal proton uptake pathway (Lee et a1. 2000). 1.7 Oxygen pathway in CcO Because of the nonpolar character of the 02 molecule, it is more concentrated in the membrane than in aqueous solution and could simply reach the active site through diffusion from the membrane into the hydrophobic regions of the protein. However, three specific oxygen pathways were proposed in the bovine enzyme based on hydrophobic channels in the crystal structure (Tsukihara et a1. 1996). In one of them, which is supported 21 by molecular dynamics studies, oxygen would reach the binuclear center through a hydrophobic hole leading from the V-shaped cleft formed by helices of subunit 111 through subunit I to the binuclear center active site (Hofacker and Schulten 1998). Based on the positions of the xenon atoms resolved in the structure of RchO crystal pre-pressurized with xenon, an alternative oxygen pathway was proposed which accessed the binuclear center through subunit I between two other transmembrane helices (Svensson-Ek et al. 2002). 1.8 The water/proton exit pathway(s) In RchO there is another channel-like structure at the interface of subunit I and subunit II. This channel is considered to be a possible water/proton exit channel. It starts from the binuclear center and continues up via the magnesium center to the outside of the membrane. The non-redox—active Mg appears to be a control point in this channel. The octahedral coordination of this metal center has 3 amino acid residues and 3 waters as its ligands (Tsukihara et al. 1996; Svensson-Ek et al. 2002). F urtherrnore, in the RchO crystal structure, water molecules are found around this region. Experiments using 180 isotopes of oxygen and water Show that water produced at the catalytic site binds to the Mg“ /Mn2+ (Schmidt et al. 2003). The evidence indicates that the Mg” could be a part of water/proton exit channel for the product released during the reaction catalysis. The results also suggested that the water produced at the active Site exits by a discrete pathway rather than by random diffusion. One possibility for the proton exit is that protons transported through the D pathway are picked up by E286. Via conformational movement of E286 and new water chain formation, 22 protons could be moved to the vicinity of the D-ring propionate group of heme a3 (Hofacker and Schulten 1998; Cukier 2004). The latter is connected to the exterior through several hydrogen-bonded water networks. However, it is not yet clear whether there is a Specific proton exit path, and if so, where. 1.9 Reaction cycle of RchO Spectroscopic and fast kinetic measurements have contributed to our understanding of the chemistry involved in 02 reduction as Shown in Figure 1.7 (Michel et al. 1998; Zaslavsky and Gennis 2000; Kim et al. 2004). The reaction cycle could start from the oxidized form ofRchO (intermediate 0), which has hydroxyls bound to both heme a3 and CuB. The first two electrons and protons that enter CcO are expected to go to €113 and heme a3 of the binuclear center, causing reduction of these two centers and the conversion of their bound hydroxyls to water (intermediate R). An oxygen can then bind to the reduced binuclear center. The oxygen bond is rapidly cleaved by a four electron transfer event, using two electrons from the heme a3 to form a ferryl-0x0 and one electron from CuB to form a cuprous hydroxide, as well as an electron and proton from a nearby Tyr288 (MacMillan et al. 1999). Intermediate P (peroxy) is formed in this step. Then a third electron and proton are injected which likely reduce the Tyr288 free radical back to its stable state, forming intermediate F. Finally, a fourth electron is injected with an accompanying proton to convert the RchO from the Fe4+ ferryl intermediate to its F e3+ oxidized state, with hydroxyls on both heme a3 and Gus. Some research results suggested that the proton pumping occurs during the oxygen reduction cycle (V ygodina et al. 1997), in the P —> F and F —> 0 steps. Others proposed that one proton is likely to have been transferred before 23 Figure l. 7: Reaction cycle of Rch0 The green circle is the CcO. The red arrows are the electron transfer pathways in Rch0, the blue arrows are the proton uptake pathways. The boxes show the reaction intermediates in the oxygen reduction chemistry. 0, oxidized; R, reduced; P, peroxy; F, ferryl. The purple circles indicate proton loading sites during transfer to the active site or to the outside. 24 the formation of the P intermediate. (Michel 1999). The proton pumping could also occur during each one-electron step of reduction of the enzyme (Sharpe and Ferguson-Miller 2008; Stuchebrukov 2007; (Wikstrom and Verkhovsky 2002; Tsukihara et al. 2003). The proton pumping mechanism and its coupling to electron transfer is still a major unsolved problem. 1.10 Proton pumping and electron transfer coupling mechanism A number of different proton pumping models have been proposed, but most are difficult to test. Proton pumping theories are generally grouped into direct coupling and indirect coupling mechanisms. However, a combination of both is also possible to explain the complicated mechanisms. In the direct coupling mechanism, the protons are bound and released by the metal centers or their direct ligands in response to changes in redox state. Since in the case of CcO there are two different types of protons involved, two separate proton uptake pathways can be proposed to bring the protons to the active Site, one for substrate protons and one for protons to be pumped. One example of direct coupling is the ligand exchange model first proposed by Chan and colleagues. In this model, a redox-active metal center, CuA, could drive the proton pump by undergoing ligand exchange, where the protonation state of the ligand changes. However, later experimental results showed that CuA, the redox center they proposed in the model, is not crucial for proton pumping (Zhen et al. 2002). Another example of a direct coupling mechanism that involves changes at the active site, is the histidine cycle model proposed first by Wikstrom and colleagues (Wikstrom et al. 1994). In this model, H290 (Rs number: H333) is proposed to cycle between imidazolium and 25 imidazolate states, so that two protons could be pumped in each of two cycles. First, H290 in the imidazolate form binds to the oxidized CUB. Then after reduction, a negatively charged oxygen intermediate binds to heme a3. To balance the negative charge, two pumped protons are taken up from the matrix side and H290 goes back to the imidazolirun state and dissociates from the CuB. At this point, two substrate protons are taken up and one water is formed. Since the electrostatic stabilization is no longer needed, the two pumped protons are released to the other side of the membrane, and H290 binds to the Cug again. The balance of electrostatics at the binuclear center is the key in the model. In a recently proposed model from our research group (Sharpe et al. 2005), the covalently bound H284—Y288 pair iS suggested to go through cycles of ligation and de-ligation from Cut; in response to changes in charge and ligand state of the CuB. The rotation of its imidazole-tyrosine rings leads to alternate opening and closing of the K pathway by breaking or reforming the tight hydrogen bond between the tyr —0H and the farnesyl-0H. Therefore, protons can move into the active site alternately from the D and K pathways, depending on the Cue ligation state and the charge present in the binuclear center. Protons are transported either to the active site to form water or to a conserved water molecule bound between the propionates of heme a3, via the histidine 334 ligand of CuB. The latter proton is released to the outside of the membrane. This is a new version of the histidine cycle model which fits with the conformational changes seen in the RchO crystal structure upon reduction (Qin et a1, 2009) but remains to be tested by other approaches. In the indirect coupling mechanism, a long-range protein-mediated coupling is required between redox changes at the active Site and conformational changes at a distance that lead to proton uptake and release. One example of the indirect coupling model was proposed by 26 Yoshikawa’s group (Yoshikawa et al. 1998). Based on their high resolution X-ray crystal structures of oxidized and reduced bovine heart CcO, residue D51 in subunit I (bovine numbering) at the top of the proposed H channel was suggested to be involved in proton pumping. The loop between subunit 1 helix 1 and helix 11, including D51, is seen to be the main region of the enzyme undergoing a conformational change. In the oxidized state of enzyme, D51 is buried under the protein surface and connected to the matrix Side via a hydrogen bonded network and internal water pools. Its conformation changes upon reduction of the CcO, leading to exposure of its protonated carboxyl to the intermembrane surface, a pK change and proton release (Yoshikawa et a1. 1998). This proton pumping mechanism bypasses the binuclear center and is proposed to be driven by heme a redox changes (Tsukihara et al. 2003). However, the residue D51 is not conserved in bacterial enzymes, so the model cannot easily be generalized. Nor is it readily testable, since mutant forms of the bovine CcO are difficult to produce and test in the purified state. 1.11 The Regulation of CcO CcO is the fourth and final complex in the respiratory chain and therefore plays a critical role in regulating the rate and efficiency of the whole energy transduction process, which impacts in turn on many physiological processes. Thus the control of CcO activity is crucial, but there are no established mechanisms for how this is accomplished. Nucleotides have been found to have regulatory effects on the CcO. In the crystal structure of bovine heart CcO two binding sites that were occupied by cholate molecules were observed and postulated to be actual binding sites for nucleotides (Shinzawa-Itoh et al. 27 2007). A deoxycholate binding Site is also found in a similar position in the RchO crystal structure, suggesting conservation and an important role for this Site (Qin et al. 2008). The activity of CcO is observed to be affected by a variety of factors, such as fatty acids (Sharpe et al. 1996), thyroid hormones (Kadenbach and Arnold 2000), zinc ions (Mills et al. 2002), nitric oxide (Antunes et al. 2004), and by the direct energy indicator, the ATP/ADP ratio (Kadenbach 1986; Beauvoit and Rigoulet 2001). The proton pumping efficiency of CcO is also regulated by changes in the membrane potential. Evidence suggests that in CcO there is a proton backflow pathway that allows protons to be taken up fiom the outside to support oxygen reduction activity in the presence of a membrane potential, decreasing the proton pumping efficiency (Mills et al. 2002). Zinc inhibition of CcO reconstituted into lipid vesicles in the presence of a membrane potential is concluded to be due to the blockage of the proton exit/backflow (Mills et al. 2002). In spite of these many possibilities, little conclusive evidence is available to provide a meaningful picture of how the activity and efficiency of CcO is regulated under physiological conditions. 2. Membrane Protein Crystallography To further understand the functional and regulatory mechanisms of the proteins, acquiring atomic-resolution structural information has become more and more important. Solving X-ray crystallographic structures is a widely used technique in the biological science field. In comparison, the nearest competing method in terms of structural analysis is nuclear magnetic resonance (NMR) spectroscopy. However, the solution-state NMR is restricted to relatively small proteins while X-ray crystallography can solve the detailed structures of relatively large proteins and membrane proteins. 28 The results from X-ray crystallography of CcO from difference sources have already provided us with a better understanding of the structure and functional relationships in this enzyme. However, there are still many questions remaining in CcO research, such as the underlying mechanism of the vectorial translocation of protons and how proton pumping is coordinated with electron transfer and oxygen reduction. Recent structures of RchO Show that not only the protein residue conformation but also the water molecules in the structure are important for the firnction (Qin et al. 2006; Qin et al. 2009). To resolve these issues, more high resolution crystal structures of CcO are still needed. Unfortunately, CcO is a large multiple-subunit transmembrane complex and high resolution X-ray crystallography Of membrane proteins has always been a challenge in the structure biology. 2.1 Challenges in membrane protein crystallography The first problem facing membrane protein crystallography is the expression and purification of the protein. To get crystals of the protein of interest, significant amounts of protein are required. Many membrane proteins exist at very low levels in the membranes natively. The over-expression of a membrane protein in the usual system, such as E. coli, often causes the protein to aggregate and be improperly posttranslationally modified. Therefore, finding a good expression system is a very important step for the membrane protein crystallography. R sphaeroides, a gram-negative bacterium with an abundant membrane system, has been proved to be a good expression host not only for CcO (Zhen et al. 1998), but also for various membrane proteins (Wadhams et al. 2000). Some evidence also shows that the lipid content in the membrane could also be critical for the expression and correct folding of the membrane proteins (Dowhan et al. 2004). In order 29 to extract a membrane protein from the lipid bilayer into solution, detergents are added to solubilize the protein. In this step, the lipid environment of the membrane protein is changed by the replacement of the lipids with detergent molecules. This usually makes the protein less stable and may lead to loss of its native conformation. Thus choosing the right detergent is very important in the crystallization of a membrane protein (1e Maire et al. 2000; Garavito and Ferguson-Miller 2001; Gohon and Popot 2003). There are three classes of detergents: ionic, nonionic, and zwitteronic, named after the biochemical features of their head groups. The nonionic detergents with sugar-based head groups, such as alkyl maltosides (Rosevear et al. 1980) are often used in membrane protein purification and crystallization since less denaturing effects on membrane proteins are observed. In our case, decyl maltoside and dodecyl maltoside were used in the solublization and crystallization of RchO (Qin et al. 2006). Zwitterionic detergents Sneh as LDAO have also been used in several successful membrane crystallization experiments (McLuskey et al. 2001). However, there are no universal standards for which detergent is the best candidate for a particular membrane protein. Successful crystallization of membrane proteins usually requires large amounts of screening for the best detergent. Another obstacle for membrane protein crystallization is the lack of proper crystal contacts for the crystallization. Membrane proteins usually have a large hydrophobic/ amphiphilic surface covered by lipids and detergents. Unlike soluble proteins, their surface lacks the hydrophilic residues which can form good ionic interactions during crystallization. Good crystals that can diffract to high resolution under X-rays usually have a well ordered formation in the crystal unit cells. The hydrophobic character of the membrane protein surface favors non-specific interactions and Often causes the protein to precipitate and 30 denature. This is one of the reasons why the right detergent is very crucial in the crystallization of membrane proteins. 2.2 Progress in membrane protein crystallization Despite the difficulties of getting membrane protein crystals, considerable progress has been made since the first reported crystal structure more than 20 years ago (Michel 1982). For example, high resolution crystal structures have been solved for CcO from various sources (Iwata et al. 1995 ; Tsukihara et a1. 1995; Tsukihara et al. 1996; Ostermeier et a1. 1997; Svensson-Ek et al. 2002; Qin et al. 2006; Qin et al. 2009). Other than high throughput screening of crystallization conditions, and availability of a variety of pure detergents and crystallization additives, other approaches have been used to overcome the difficulty of membrane protein crystallization. These approaches include micro- and macro-seeding (Fromme and Witt 1998), semi-directed mutagenesis of selected surface residues (Pautsch et al. 1999) and crystal dehydration (Kuo et a1. 2003). Besides the conventional crystallization methods, several new techniques involving use of lipids and adding protein domains have been developed to aid in membrane protein crystallization. 2.2.1 Crystallization of membrane proteins in lipid cubic phase The lipid cubic phase methods were first developed in hope of maintaining the native environment of the protein including the lateral pressure exerted by lipid bilayers and minimizing the disturbance and disordering of the membrane protein. In this method, a continuous membrane formed by mixing lipids and water is used as a support matrix for nucleation and crystal growth. Since membrane proteins tend to be less stable when they are extracted from native membranes and incorporated into detergent micelles, 31 crystallizing membrane proteins in a quasi-solid membrane environment throughout the crystallization process seems to have an advantage. The first successful case for lipid cubic phase crystallization was the application to bacteriorhodopsin (Landau and Rosenbusch 1996). Later results Showed that this method was very successful in a class of membrane proteins with seven transmembrane helices including bacteriorhodopsin (Landau and Rosenbusch 1996; Luecke et al. 1999), halorhodopsin (Kolbe et al. 2000), and sensory rhodopsin ll (Gordeliy et al. 2002). 2.2.2 Antibody assisted membrane protein crystallization One of the main reasons membrane proteins are difficult to crystallize is their lack of hydrophilic residues on the protein surface, which are important in forming crystal contacts between molecules. Adding certain peptides or proteins, such as an antibody, could extend the hydrophilic portion of the membrane protein, thus favoring more crystal contacts. In the crystal structure of the four subunit cytochrome c oxidase from Paracoccus denitrificans (Pd), all crystal contacts were found between attached antibody molecules (Iwata et al. 1995). The antibody assisted cryStallization also succeeded in the crystallization of the two subunit PdCcO (Ostermeier et al. 1997), yeast cytochrome bcl complex (Hunte et al. 2000), and the KcsA K+ channel (Zhou et al. 2001). 2.2.3 Fusion protein strategy to assist membrane protein crystallization Another way of increasing the hydrophilic portion of a membrane protein is fusion to a soluble protein. One example of this method is the crystallization of E. coli cytochrome b03 ubiquinol oxidase, in which case a small peptide (peptide Z) was engineered into the membrane protein with the hope of forming crystal contacts (Byme et al. 2000). However, the fusion protein approach did not produced well-diffracting crystals yet, presumably 32 because of the flexible nature of the linker region. One successful case of the fusion protein strategy is the solving of the first crystal structure of human B2-adrenergic G protein-coupled receptor (GPCR) (Cherezov et al. 2007). In this case, Cherezov et al. removed the flexible loop of the GPCR and inserted a T4-lysozyme by molecular engineering. Combined with the cubic phase method, the firsion protein was crystallized and diffracted to 2.4 A. The widely-used bio-fluorescent marker protein, green fluorescent protein (GFP), could also be used in the fusion protein strategy. The highly soluble, compact structure of the GFP makes it a good candidate in the crystallization of various proteins (Hakansson and Winther 2007; Suzuki et al. 2010). 3. Background of Green Fluorescent Protein The green fluorescent protein (GFP, as shown in Figure 1.8) is a spontaneously fluorescent soluble protein isolated from the jellyfish, Aequoria Victoria (Shimomura et al. 1962; Ormo et al. 1996; Tsien 1998). Its natural role is to transduce the blue chemiluminescence of aequorin into green fluorescent light by energy transfer (reference). However, the purpose of both bioluminescence and GFP fluorescence in jellyfish is unknown. The structure and fluorescent mechanisms of GFP have been well studied since it was first found by Shimomura et al in 1960 (Shimomura et al. 1962). GFP has become a good expression marker that is widely used in life science research. Roger Tsien, Martin Chalfie and Osarnu Shimomura were awarded the 2008 Nobel Prize in chemistry for their discovery and the development of the GFP. 33 Figure 1. 8: Structure of Green Fluorescent Protein. GFP is composed of 11 B-sheets and 4 a-helices. The chromophore (yellow colored structure) is buried inside this B-Sheet barrel structure. This figure is created by ViewLite software with coordinates from PDB file lEMC. 34 3.1 Structure of GFP WT GFP is composed of 238 amino acid residues (MW 26.9 kDa). It has a typical beta barrel structure, consisting of 11 B-Sheets with one alpha helix structure containing the chromophore in the center (Orrnoe et al. 1996). The chromophore of GFP is a p-hydroxybenzylideneimidazolinone. The Side chains of the barrel face inward and induce Specific cyclization reactions in the conserved tripeptide Ser65—Tyr66—Gly67 that lead to chromophore formation. Figure 1.9 shows the current proposed mechanism for chromophore formation (Heim et al. 1994; Cubitt et al. 1995). GFP first folds into a near-native conformation, then the nucleophilic attack of the amide of Gly67 on the carbonyl of Ser65 forms the imidazolinone structure. After a dehydration reaction, molecular oxygen dehydrogenates the (l—B bond of residue 66 to put its aromatic group into conjugation with the imidazolinone. This process is not dependent on other proteins or special cofactors except oxygen. The formation of the chromophore does not happen in obligate anaerobes. Oxygen has no further effect after the formation of the chromophore (Swaminathan et a1. 1997) . 3.2 GFP derivatives The hydrogen bonding network and electron stacking interactions with side chains near the chromophore influence the fluorescent properties of GF P. Therefore many different mutants of GFP have been engineered for the potential of widespread usage and the evolving needs of researchers. A Single mutant (S65T) reported in 1995 in Nature by Roger Tsien was the first major improvement on WT GFP. It greatly improved the spectral characteristics of GFP, resulting in increased fluorescence and photostability. The major 35 R Tyl‘66 0 Gly67 or other unfolded/denatured conformation Ser or Thrc65 (R = H or Me) OH folding (t1 ;2 ~10 min) 0 N/fil HN OH 0 . R ~N N H dehydration l H tu2 ~ 3 min for OH * 0H cyclization + dehydration aerial oxidation 0 02 113:2? ’ \N/WI/ \N O/\“ ' O R t,,2~I9H 83min HO N H OH OH :2 Figure l. 9: A proposed mechanism of the formation of the chromosphere of GFP. In this mechanism (Heim et al. 1994; Cubitt et al. 1995), the formation of the chromophore is a self-catalyzed reaction and does not require other protein factors. 36 excitation peak of this mutant is 488 nm and the peak emission stays at 509 nm. This matched the spectral characteristics of commonly available FITC filter sets, increasing the practicality of use by the general researcher. Many other color mutations have also been made, such as blue fluorescent protein (EBF P), cyan fluorescent protein (ECFP) and yellow fluorescent protein derivatives (EYFP). EBF P derivatives contain the Y66H mutation, which causes the chromophore to form with an indole rather than phenol component. Several additional compensatory mutations in the surrounding barrel are required to restore brightness to this modified chromophore due to the increased bulk of the indole group. The red-shifted wavelength of the EYFP derivatives is accomplished by the T203Y mutation and is due to rt-electron stacking interactions between the substituted tyrosine residue and the chromophore. Besides the color variation, some other modifications have also been made on GFP to change its fluorescent characteristics, such as calcium-sensitive GFP, redox-sensitive GFP and pH-sensitive GFP. 3.3 Usage of GFP and its derivatives OF P and its derivatives are widely used in the life science research field. OF P is usually much less harmful when illuminated in living cells compared to most small fluorescent molecules such as fluorescein isothiocyanate and FITC, which are strongly phototoxic when used in live cells. GFP can also be expressed in many different organisms, from bacteria to mammalian cells, without losing its fluorescence properties. This makes OF P a very good expression marker for detecting gene expression and regulation. The emergence of C32+-SCI‘IS111VC, pH-sensitive (Miesenbock et al. 1998) and redox-sensitive GFP derivatives (Hanson et al. 2004; Cannon and Remington 2006) also allows observation and 37 study of changes of physiological conditions in living cells or various systems, such as reconstituted vesicles. Another important use of GFP is the application in the fluorescence resonance energy transfer (FRET). An electronic excited donor chromophore may transfer energy to a proximate acceptor chromophore through nonradiative dipole—dipole coupling. By this mechanism, FRET can be used to investigate the interaction between macromolecules and the dynamic molecular events within living cells (Pollok and Heim 1999). Besides the fluorescence applications of GP P, it could also be used as a fusion protein tag for crystallization. In the crystallization of GFP-ubiquitin and GFP-UBM firsion proteins, the tyrosine residues that are involved in mediating crystal contacts are arranged in a belt on the surface of the [3 -barrel structure of GFP in both crystals (Hakansson and Winther 2007; Suzuki et al. 2010). This indicated that OF P could assist the crystallization of fusion proteins. Considering its good solubility and structural stability, GFP is a good candidate for a fusion partner to increase the hydrophilic portion of a membrane protein. 38 CHAPTER 2. MATERIAL AND METHODS 1. Molecular Engineering of Rhodobacter sphaeroides Molecular engineering has been performed on different Rhodobacter sphaeroides strains to produce various mutants of RchO. Histidine tags are engineered onto either the C-terminus of subunit I or the C-terminus of subunit II of RchO (Zhen et a1, 1998) so that after expression the protein could be purified with Ni-NTA columns. Genes encoding all four subunits were inserted into the over-expression plasmid pRK415-1 (Zhen et al. 1998) in different combinations. Biparental conjugation (Zhen et al., 1998) was used to transfer the expression plasmid from E. coli strain S17-1 to Rhodobacter expression host strains (A123 or Al A4). Different constructs of RchO used in this research and their genotyes are listed in Table 2.1. Detailed constructions of the expression plasmids for these mutants are Shown in each chapter. 2. Cell Growth Rhodobacter sphaeroides strains were first streaked from single colonies onto Sistrom’s plates and grown at 30°C for 4-5 days. Then the cells were scraped into 2.8 L Fembach flasks containing 800 mL of Sistrom’s medium (Sistrom et a1, 1962) with 25mg/mL streptomycin, 25 mg/mL Spectinomycin and 1mg/mL tetracycline and grown aerobically in the dark at 30°C as described (Hiser et al. 2001). After 3 days at an optical density of 1.5-1.8 at 600 nm and pH of 8.5-9.0, R. sphaeroides cells were harvested by centrifugation in a GS-3 rotor at 14,000 x g for 20 minutes at 4°C. The KPi buffer 39 . Host Hista StraIn name strain positifin Phenotyes PJL16 A123 Subunit II Subunit I —EYFP fusion mutant, wild type (Chapter 3) subunit IV EYF P gene is linked through a 4 amino acids linker (GGAS) to the C-terminus of subunit 1. PJL33 A123 Subunit II Short-Short subunit I mutant. (Chapter 4) 16 amino acids were removed from the C-terminus of subunit 1. PJL45 A123 Subunit I E552A mutation on subunit 1. wild type (Chapter 4) subunit IV PJ L49 A123 Subunit 1 Short subunit I mutant. Wild type subunit IV (Chapter 4) W560 is mutated into a stop codon (TGG->TGA), so last 6 amino acid of subunit I C-terminus are “deleted” from the expressed protein. PJL63 A123 Subunit II Subunit 1 —subunit III fusion mutant. Wild (Chapter 4) type subunit IV. N-terminus of subunit III gene is linked to the C-terminus of subunit 1. PJ L34 A1A4 Subunit II K362M with short Short subunit IV (Chapter 5) PJL35 A1A4 Subunit II D132A with short Short subunit IV (Chapter 5) Table 2. l: Mutant strains created and used in this thesis 40 containing 50 mM KH2PO4 and 1 mM EDTA, pH 6.5 (Zhen et al. 1998) was used to resuspend the harvested cells. The cells were stored frozen at -70°C. 3. Preparation of R.sphaeroides Cytoplasmic Membranes Before the thawing of Rsphaeroides cells , small amounts of DNase I and RNase were added. More KPi buffer was added according to the amount of the cells (for a growth of 24L the final volume of the cell resuspension is usually ~ 200 mL). The cell resuspension was then homogenized and cells were broken with two passages through the French‘press at 20,000 psi. Unbroken cells and debris were removed by centrifugation at 30,000 x g for 30 minutes at 4°C. Then the supernatant was collected and an ultracentrifugation at 200,000 x g for 1.5 hours was performed to harvest the cell membrane fraction. The cell membrane pellet was resuspended in either a lower salt buffer containing 10 mM Tris, 40 mM KCl, pH 8.0 for subunit I histidine-tagged strains, or a high salt buffer containing 10 mM Tris, 220 mM KCl, pH 8.0 for the subunit II histidine-tagged strains. The membrane sample were then quick-frozen in liquid nitrogen and stored at -80°C. 4. UV-Visible Spectroscopy UV-Visible Spectra were taken using a Perkin-Elmer Lambda 4OP UV-visible spectrophotometer. A reduced minus oxidized CcO difference Spectrum was performed on the R. sphaeroides membrane fraction; sodium dithionite and fenicyanide were used to fully reduce or oxidize the enzyme samples, respectively. The enzyme was first diluted in a buffer containing 100 mM HEPES, pH 7.9, 1 mM EDTA, and 0.1% dodecyl maltoside prior to the spectra being taken. The extinction coefficient used was 45606-630 = 24 41 cm'lmM'l (Zhen et al. 1998). For absolute Spectra of the dithionite-reduced CcO, the extinction coefficient used was A 8 606-640 = 40 cm'lmM'I (Zhen et al. 1998). 5. Protein Concentration Assay The BCA (bicinichonic acid) Protein Assay Reagent Kit from Pierce (Smith et a1. 1985) was used to measure the total protein concentration in the membrane sample. Bovine serum albumin (BSA) standards and the resuspended membrane samples were diluted in a buffer containing 10 mM Tris, 40 mM KCl and 0.25% deoxycholate, (pH 11.2). Reagent A containing 1% BCA-Na2, 2% Na2CO3.H2O, 0.16% Na2 tartrate, 0.4% NaOH, 0.95% NaHCO3, pH 11.25 and reagent B containing 4% CuSO4-5 H2O were mixed in a 50:1 ratio, and then 2 mL of the mixture was added to each of the diluted sample and BSA standard tubes. After incubation at 60°C for 30 min, the absorbance of these samples was measured at 562 nm. A standard curve was produced with the absorbance measurements at 562 nm for the BSA standards with known protein concentration. The protein concentration of the membrane sample was calculated with this standard curve. 6. Solublization of the Membranes The R. sphaeroides. cell membrane sample was diluted to a protein concentration of 10 mg/ml with buffer containing 10 mM Tris (pH 8.0), 40 mM or 220 mM KCl depending on the position of the histidine tag as described above, and 1 mM imidazole. Dodecyl 'maltoside was then added to the membrane resuspension sample to a final concentration of 1% (w/v). The solution was stirred for 20 minutes at 4°C and unsolublized material was 42 removed by ultracentrifugation at 200,000 x g for 30 minutes. The supernatant containing Rch0 was collected and used immediately for further column purification. 7. SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect the CcO subunit composition including different forms of subunits and the impurities contained in the CcO sample. Approximately 10 pg of protein in sample buffer containing 25 mM Tris, pH 6.5, 40% glycerol, 8% SDS and 0.08% bromophenol blue was loaded onto an 8% acrylamide stacking gel at pH 6.8 on top of an 18% acrylamide separating gel at pH 8.8. The gel was electrophoresed at 100 V at room temperature (Peiffer et al. 1990). The gel was then stained with Coomassie blue and destained with 7.5% glacial acetic acid for 3 times (Hiser et al. 2001). Polypeptide sizes were estimated from low-molecular weight range markers from New England Biolabs. 8. Purification of the RchO In order to obtain purified RchO enzyme complex for crystallization or the reconstituted vesicle assay, different FPLC (fast protein liquid chromatography) column purification protocols were used. For crystallization, the enzyme was purified via a single step Ni-NTA FPLC purification method developed by Ling Qin (Qin et al. 2006). To reconstitute RchO into vesicles, a further purification procedure with a DEAE sepharose ion-exchange column was used. 43 8. 1 Ni-NTA purification of the Subunit I Histidine-Tagged RchO A home-packed Ni-NTA (Qiagen) column (typical volume: 18 ml) connected to an AKTA FPLC system (Pharmacia) was used to purify the RchO with histidine—tags on the C-terminus of subunit 1. The column was first equilibrated with 80 m1 of low salt buffer A containing 10 mM Tris, 40 mM KCl, 10 mM imidazole and 0.05% dodecyl maltoside, pH 8.0. The solubilized membrane fraction of Rch0 was Slowly loaded onto the column via a 150 mL superloop (flow speed 0.4 mL/min). The column was washed extensively with 12 column volumes of buffer A. A linear gradient of 0 - 100% of 15 column volumes of buffer B. containing 10 mM Tris (pH 8.0), 40 mM KCl, 150 mM imidazole, 0.05% dodecyl maltoside was applied onto the column after the washing step. The RchO containing fractions were selected based upon the UV absorbance from the FPLC and then collected together. The imidazole in the collected fraction was removed by washing and concentrating multiple times by using an Amicon YM100 filter unit (MW cutoff 100 kDa; maximum volume 15ml, Millipore). The yield of this column purificatiOn step was typically around 50%. 8.2 Ni-NTA Purification of the Subunit II Histidine-Tagged 'RSCCO The method used for the subunit II histidine tagged RchO purification is very similar to the method for the subunit I His-tag RchO. Firstly the detergent solublized membrane sample was loaded onto the home-packed Ni-NTA column (column volume: 18-20 ml) connected to an AKTA FPLC system that was pre-equilibrated with 80 ml high salt buffer A containing 10 mM Tris , 220 mM KCl, 2.5 mM imidazole and 0.05% dodecyl maltoside, pH 8.0. After washing with 12 column volumes of buffer A, a two step gradient of increasing concentration of buffer B containing 10 mM Tris, pH 8.0, 220 mM KCl, 150 44 mM imidazole, 0.05% dodecyl maltoside was used instead of the one step gradient step in methods 2.8.1. First the percentage of buffer B was increased to approximately 8-10% (approximate imidazole concentration: 15 mM) and held constant for approximately 6 column volumes. The first peak, a impure, red-colored cytochrome c oxidase bound to an contaminating Species including cytochrome bcl was eluted in this step. After this washing step, the concentration of buffer B was increased to 100% (final imidazole concentration: 150 mM) with a linear gradient and the second peak containing purer green colored CcO was eluted and the green fractions in the second peak were collected. The collected enzyme was then concentrated with a Amicon YM100 filter unit. The buffer was exchanged to 10 mM Tris, pH 8.0, 150 mM NaCl, with 0.16% decyl maltoside by washing and concentrating 2 — 3 times. The typical yield of purified protein is approximately 30-40%. 8. 3 Ion-exchange Column Purification of RchO For RchO that is used for activity and proton pumping mechanism study, a second purification with DEAE ion exchange column was performed. Concentrated enzyme fi'om Ni-NTA purification (equilibrated in buffer A below) was loaded onto a DEAE ion exchange column (column Size: 20 ml) pre-equilibrated with 100 ml buffer A containing 10 mM Tris, 0.05% dodecyl maltoside, pH 8.0. The column was washed with buffer A for three column volumes before a linear gradient of 0-100% buffer B containing 10 mM Tris, 0.5 M KCl, 0.05% dodecyl maltoside, pH 8.0 was applied. The length of the gradient was 15 column volumes. Green fractions from the linear gradient were pooled, concentrated and the buffer was exchanged to 10 mM Tris, pH 8.0, 0.16% - 0.24% decyl maltoside by washing and concentrating 2 - 3 times by using a YM100 filter unit or in a stirred 45 ultrafiltration cell as described above. The typical yield of this column purification step with washing and concentrating was around 50%. 9. Reconstitution of Cytochrome c Oxidase into Vesicles To investigate proton pumping abilities and the respiratory control ratios of the various RchO proteins, the Rch0 was reconstituted into vesicles (RsCOV). A cholate dialysis method (Hiser, Mills et al. 2001) is used to reconstitute RsCOVs. In this method, cholate detergent is added to the enzyme to displace the dodecyl/decyl maltoside detergent from the enzyme. Cholate detergent is subsequently easily eliminated by dialysis due to its high CMC, critical micelle concentration, of 14 mM. As the detergent concentration is gradually decreased in the presence of lipid a unilamellar vesicular strricture is formed. The soybean polar lipid extract, asolectin obtained from Avanti Polar Lipid, Inc. (Cat No 541602) is used in the procedure. It mainly contains phosphatidyl ethanolamine (22.1%), phosphatidyl inositol (18.4%), phosphatidyl choline (45.7%), phosphatidic acid (6.9%), and other components (6.9%). Before usage, the asolectin was precipitated with acetone and then extracted with diethyl ether. The asolectin was suspended to a concentration of 40mg/m1 and sonicated at 4°C under argon gas using a microtip sonicator (Heat systems-Ultrasonics sonicator Model W-225) with in a buffer containing 75 mM HEPES-KOH, 14 mM KCl (pH 7.4) 2% cholate. After the solution became translucent, the suspension was centrifuged for 15min at 12,000 x g to remove unsolublized particles. Rch0 was prepared at a final concentration of 4uM with 4% cholate in the same buffer condition (75 mM HEPES-KOH, 14 mM KCl, pH7.4). The RchO solution was then mixed with the sonicated asolectin suspension to obtain a final concentration of 2 pM 46 oxidase and 20 mg/ml lipids (Hiser et al. 2001). The cholate was removed using five steps of dialysis with rapid stirring at 4°C in Spectrapor dialysis tubing (#25225/204, 12-14,000 Mr cut-off) to remove detergent and form the COVS. (1) 6 hours in a 100 volume of 75mM HEPES-KOH pH7.4, 14mM KCl with, and (2) without 0.1% cholate respectively; (3) 12 hours in 100 voltunes of 50 mM HEPES-KOH pH7.4, 25mM KCl, 15 mM sucrose; (4) and (5) a 12 hours dialysis two times in 500 volume of 50 M HEPES-KOH (pH 7.4), 45 mM KCl and 44 mM sucrose as previously described (Hosler et al. 1992). Sucrose was added to maintain the osmotic conditions. After cholate dialysis, the vesicles were shown to have CcO in the predominantly mitochondrial configuration (N icholls et al. 1980) with the the cytochrome c binding Site on the outside and to have a high respiratory control ratio. 10. Cytochrome c Oxidase Activity Assay The oxidase activity of Rch0 and was measured polarographically by using a Gilson model 5/6H oxygraph with a closed therrnostatted chamber containing a Clark-type oxygen electrode (F erguson-Miller et al. 1976). The assay is performed at 25°C in a 1.75 mL reaction cell containing 50 mM KH2PO4, pH 6.5, 0.05% dodecyl maltoside, 2.8 mM ascorbate, 0.55 mM TMPD [and 30 1.1M of horse heart cytochrome c. The oxidase concentrations were in the range of 5 nM to 50 nM. Soybean phospholipids, asolectin (1 mg/mL) was also added to stimulate the enzyme for full activity. The activity of RchO is calculated as described in (Fetter et al. 1995) Measurement of the activity of COVS was performed in the controlled state (absence of ionophores), and with the addition Of valinomycin, which dissipates the membrane 47 potential by equilibrating Klr across the membrane to remove the charge (Aw=0). The uncontrolled condition occurs with the addition of uncouplers, FCCP (or CCCP), which transfers protons across the membrane, and this causes the proton gradient to be dissipated (ApH=0) and shows the highest rate of activity. Then the Respiratory Control Ratio, RCR for the RsCOV is calculated (RCR=uncontrolled state activity/ controlled state activity). For a typical cytochrome c oxidation rate measurement, 4.2 uM of cyt2+ was mixed with 0.04 pM of M3 in the COVS (Inside buffer: 50 mM HEPES-KOH, pH7.4 +43 mM KCl). Data scans were collected (1000 scans/s) by Photophysics stopped flow apparatus. For the measurement of alkalinization inside the vesicle, 4.2 uM of cytochrome c was mixed with 0.04 uM of oxidase in the COVS (Inside buffer: 50 mM HEPES-KOH, pH 7.4 +43 mM KCl). Data scans were collected (1000 scans/S) by Photophysics stopped-flow apparatus. For EYFP-COVS, the fluorescence is and detected at 525 nm. For the wild type COVS, the fluorescence is excited at 465 nm detected at 510 nm 11. Crystallization of the RchO 11.1 Four-subunit RchO Crystallization To obtain the four-subunit RchO crystals, either hanging drop or sitting drop crystallization setups could be used. Figure 2.1 shows the hanging-drop crystallization set-up and Sitting-drop crystallization setup. In the hanging drop set up, the crystal grows faster than the sitting drop set up. However, in a sitting-drop crystallization experiment, a much larger volume can be used in order to obtain larger crystals. All the crystallization experiments were performed at 4°C. In the hanging-drop crystallization set up, 2 pL of enzyme solution containing 10 mM Tris, 0.16-0.24% decyl maltoside, pH 8.0 and 100-120 48 Coverslip Protein solution Reservoir solution A: Hanging Drop Sealing tape Protein solution Reservoir solution B: Sitting Drop Figure 2. l: Crystallization setup for the Rch0 49 uM CcO was mixed with 1 ILL of reservoir solution containing 100 mM sodium citrate, 100 mM NaCl, 18-23% PEG-400, pH 5.6-5.8 and 1 uL of crystallization additive solution containing 5% heptanetriol, 33 mM MgCl2, and 0.026% dodelcyl maltoside. The crystallization drop was then incubated at 4°C for crystal growing. Triangular crystals of four subunit RchO usually appeared after 3-4 days and continued growing to up to 0.2 x 0.2 x 0.1 mm in approximately two weeks. In the sitting drOp crystallization set-up, the typical volume of the drop solution increased to 6 HL of enzyme solution containing 10 mM Tris, pH 8.0, 0.16-0.20% decyl maltoside and 80-120 uM CcO mixed with 3 ILL of reservoir solution and 3 ILL of crystallization additive solution as described above. The crystallization drop was incubated at 4°C and triangular crystals usually appears after 2 weeks incubation. 11.2 Two-subunit RchO Crystallization In a typical crystallization experiment to obtain crystals of the [-11 subtmit RchO, a sitting drop crystallization set-up was mainly used as Shown in Figure 2.2. For a typical crystallization set-up, 6 uL of enzyme solution containing 10 mM Tris, 50 mM NaCl, 0.20% decyl maltoside, pH 8.0 and 120 uM CcO was mixed with 3 1.1L of reservoir solution containing 100 mM MES, pH 6.3-6.9, 24-26% PEG-400 and 3 11L of crystallization additive solution containing 5% heptanetriol, 32 mM MgCl2, 1.3 mM CdCl2 and 0.026% dodecyl maltoside. The crystallization drop was then incubated at 4°C and crystal showers of tiny triangular crystals of the four subunit RchO started to appear after approximately 3-4 days. Larger, clustered crystals of I-II subunit RchO crystals started to appear after 50 approximately 2 weeks and continued to grow to their full size of up to 0.2 x 0.2 x 0.1 mm in 3-4 weeks. 12. Flashcooling of the RchO Crystals F lashcooling of the crystals was performed to freeze the crystals prior the synchrotron X-ray diffraction. The 4-subunit RchO crystals were grown at 4°C, and would quickly dissolve under room temperature, thus the flashcooling procedures were performed in the cold room. Drierite could be used as a desiccant in the cold room to reduced the humidity and prevent the formation of ice crystals during the procedure. The crystals of the RchO were picked up from the original crystallization drop with a cryoloop and soaked in approximately 25 uL of stabilizing solution in a sitting drop well. The stabilizing solution was made to mimic the assumed concentration of ingredients in the crystallization drop after equilibrium except for a little less detergent in a sitting drop well. Usually the stabilizing solution contained 91 mM sodium citrate (pH 5.6-5.8), 91 mM NaCl, 18 mM Tris (pH 8.0), 30 mM MgCl2, 1.5 mM CdCl, 2.5% heptanetriol, 0.16% decyl maltoside, 0.016% dodecyl maltoside, and 18-23% PEG-400. A small aliquot (IOuL) of the cryosolution containing the same ingredients as those in the stabilizing solution except for a higher (30-32%) PEG-400 concentration was added to the stabilizing solution containing the crystals and then the two solutions were carefully mixed. A small aliquot (10 uL) was then taken out from the sitting drOp well to return the drop volume to 30 pl. The same procedure was repeated several times until approximately the entire solution in the Sitting drop well had been exchanged to the cryosolution. Adding small aliquots of the cryosolution ensured the gradual increase of PEG-400 concentration and thus protected the crystals from being damaged due to dramatic increase in the osmotic pressure in the 51 surrounding environments. The crystals were then picked up again using a cryoloop and submerged into liquid nitrogen. The flashcooled crystals were then stored in liquid nitrogen in cryovials until data collection. 13. Data Collection and Processing X-ray diffraction data were collected at GM/CA-CAT Station 23 D-B and LS-CAT, Station 23D-G, Advanced Photon Sources, Argonne National Laboratory. The detector used was MARCCD-300. The data set was collected by the rotation method. During the data collection from the HI subunit CcO crystals, a 180° rotation about the it angle was performed with 0.3° per frame. The exposure time was set to 0.5second to 2 second depended on the X-ray intensity. Sometimes two data sets were collected on a single crystal, one for the high resolution range with a crystal-to-detector distance of about 180 mm and a longer X-ray exposure time, and another data set for the low resolution range with a crystal-to-detector distance of approximately 260 mm and a shorter X-ray exposure time. The data were integrated, scaled and merged using with the software HKL2000 (Otwinowski and Minor 1997). 14. Molecular Replacement and Structural Refinement For the crystal structure determination of the four subunit RchO, a rigid body refinement using the coordinates of the published structure of the four subunit RchO (PDB entry 1M56) or structure of the 2 subunit RchO (2GSM) as the initial phasing model was performed with CNS 1.1 (Brunger et al. 1998), followed by simulated annealing, and cycles of energy minimization, model visualization and manual model building. A group B 52 factor refinement was performed at later stages of the refinement. Molecular visualization and model building were performed by using the program (Sack and Quiocho 1997). For the crystal structure determination of the I-11 subunit RchO, an initial molecular replacement search was performed by using the program Phaser (Storoni et al. 2004), using the coordinates of the two-subunit cytochrome c oxidase (PDB entry 2GSIVD as the original search model. Refinement of the structure was performed with CNS 1.1 using cycles of simulated annealing, and energy minimization (Brunger et al. 1998). B factors were refined at the later stages. Molecular visualization and model building were performed by using the program CHAIN (Sack and Quiocho 1997). The structure was first refined at 2.5 A resolution and then the resolution was extended to include all data up to 2.15 A. Towards the end of refinement, restrained refinement with TLS refinement were performed using Refmac5 (Bailey 1994; Murshudov et al. 1997; Winn et a1. 2001). 53 CHAPTER 3. CONSTRUCTION OF A FUSION PROTEIN OF RSCCO WITH A GREEN FLUORESCENT PROTEIN VARIANT FOR CRYSTALLOGRAPHY AND PROTON PUMPING MECHANISM STUDY 1. Introduction Resolution of the structure of CcO is a crucial requirement for the full elucidation of the function and mechanism. Unfortunately, membrane proteins, due to their amphiphilic character, are particularly difficult to crystallize, and success in growing three-dimensional crystals is relatively infrequent. For the RchO, more high-resolution crystal structures are still needed, especially of key mutants of CcO. Protein engineering provides new hope to solve this problem. Analysis of the crystal packing of the available membrane proteins shows that most crystal lattice contacts are made by the polar extra-membranous parts of the membrane protein. Therefore, increasing the polar part of a membrane protein could be a promising method of getting a well-ordered membrane protein crystal. This could be achieved by adding the proper antibody, as was done for the crystallization of CcO from Paracoccus denitrificans (Iwata et a1. 1995). Another method is to use genetic engineering to fuse a soluble protein to the polar part of the membrane protein. Green fluorescent protein, GFP, is a good choice for making this kind of fusion protein(Hakansson and Winther 2007; Suzuki et al. 2010). 54 OF P is a spontaneously-fluorescent soluble protein isolated from the jellyfish, Aequoria victoria (Figure 1.8). Its natural role is to transduce the blue chemiluminescence of another protein, aequorin, into green fluorescent light by energy transfer. Its crystal structure and mechanism of fluorescence have been well studied. The formation Of this chromophore is not dependent on other proteins or special co-factors. Thus active GFP can be expressed in many species, from bacteria to mammalian cells. The special chemical and physical characteristics of GFP also make it an ideal fusion protein tag. GFP is very stable under physiological conditions. Denaturation of GFP requires treatment with 6 M guanidine hydrochloride at 90°C or pH of <40 or >12.0. Many GFP mutants have been produced. An interesting member of them is a yellow colored pH sensitive GFP, named Enhanced Yellow Fluorescent Protein or EYFP (Elsliger et al. 1999). The fluorescence intensity of this mutant, which is modified in residues T203Y/S6SGN68L/S72A, changes according to the environmental pH, so that it can be used as an intracellular pH indicator. In our case, it could be used with CcO reconstituted into vesicles to study proton pumping. Thus, in addition to providing a possible route to a higher resolution structure, a CcO-EYFP fusion protein could provide a sensitive tool for monitoring CcO function. 2. Methods and Results The purpose of making CcO-GF P fusion proteins is to increase the hydrophilic portion of this membrane protein complex. By doing this the CcO molecules could have better contacts with each other and form better crystals that diffract to higher resolution. A GFP 55 Normalized Fluorescence Figure 3. l: GFP pH sensitive mutants. This chart Shows that the fluorescence strength of these mutant proteins is correlated to the pH change ( A: EYFP. III: EYFP-H148Q ). (Elsliger, M et a1 Biochemistry 38, 5296-5301. 1999 ) 56 mutant, EYFP, was used in this project (gift from James Remington, University of Oregon). The fluorescence strength of EYFP is better than that of the traditional GFP and its pH sensitivity make it doubly valuable. In the pH range from 6 to 8, its fluorescence emission will increase with an increase in pH (Figure 3.1). A R. sphaeroides strain A123(Zhen et al. 1998) was chosen as the expression host strain. The genes for CcO subunits I, II and III are deleted in this strain to provide a clean background for the expression of CcO-GFP fusion proteins. 2.1 Expression of GFP under the control of CcO subunit I promoter in Rs As the first step, to prove that EYFP can be expressed in Rs, an EYFP expression plasmid, pJL-4, was constructed (Figure 3.2). A Splicing PCR method was used to integrate the gene of subunit 1 promoter and the coding region of EYFP together. A histidine tag and an EK site were placed between the CcO subunit I promoter and the EYFP gene so that the expressed protein could be firrther purified by Ni-NTA column chromatography. This fragment was cut with PstI and ligated into the multi-cloning site of the vector pRK415. The pRK415 is a widely used expression vector for R. sphaeroides (Zhen et al. 1998). It can be maintained in E. coli cells, and transferred into R. sphaeroides cells by conjugation. Firstly, through transformation, pJL-4 was transferred into E.coli strain 817-]. Then R. sphaeroides strain A123 cells were mixed with these 817-] cells. In this way, pJL-4 was transferred into R. sphaeroides cells through conjugation(Zhen et al. 1998). Finally, the positive colonies were picked up on tetracycline-containing Sistrom plates and named as PJL4. A R. sphaeroides negative control strain PJL-C was constructed with the Similar procedures except that a pRK415 empty vector was used as the conjugation plasmid. 57 Multiple cloning site EK site EYFP gene PstI PstI Figure 3. 2: Plasmid construction of pJL-4. The gene for EYFP (yellow) was PCR cloned and ligated behind the CcO subunit I promoter (blue). Between the promoter and the EYFP gene, a DNA fragment containing a encoding 6 x histidine tag and a EK site were inserted. The histidine tag can be used to purify the expressed protein with a Ni-NTA column. The EK site is a proteolysis site that can be used to remove the His-tag from the purified protein. The whole fragment was then ligated into the pRK415-1 plasmid at the Pst I site. 58 EYFP Signal expressed 30KD —>- -- in R. sphaeroides Figure 3. 3: Expression of EYFP in Rs. Western blot samples: Lane 1: OF P protein (expressed and purified from E. coli); Lane 2: EYFP protein (expressed and purified from E. coli); Lane 3: Cell lysates of JL-C strain (negative control); Lane 4: Cell lysates of J L4 strain. SDS-PAGE gel concentration: 12% 59 Protein expression in R. sphaeroides was detected by western blotting using a OF P Specific monoclonal antibody. Purified OF P and EYFP proteins (produced in E. coli) were used as the positive controls. R. sphaeroides strain cell lysates were prepared and the proteins are separated by SDS-PAGE. The results were Shown in Figure 3.3, Showing that this monoclonal GFP antibody can be used to detect both GFP and EYFP proteins. An EYFP Signal was also detected in the cell lysates of the PJL4 strain, indicating that the EYFP was successfirlly expressed in this R. sphaeroides strain. 2.2 Expression of the CcO-EYFP fusion protein in R. sphaeroides A CcO subunit 1- EYFP expression plasmid pJL-16 was constructed (Figure 3.4). It contains the subunit I-EYFP fusion sequence and a His-tagged subunit II in its operon withe the wild type III operon. The whole protein complex can be purified with a Ni-NTA column. A short linker peptide sequence, GGAS, was placed between subunit I and EYFP to avoid a possible folding disruption. pJL-16 was transferred into R. sphaeroides strain A123 by conjugation to produce the CcO-EYF P expression strain PJL16. The expression of COO-EYFP was examined by both spectrophotometric analysis and western blotting (Figure 3.5). The western blot showed that the subunit I-EYFP fusion protein was expressed in JL16 cells and most of the signal was concentrated in the membrane fraction (Figure 3.5A). Reduced forms of heme a and heme a3 in CcO Should have a specific absorbance peak at 606 nm. Thus the CcO in the membrane extraction of R. sphaeroides can be identified and quantified by the dithionite-reduced minus ferricynide-oxidized difference spectrum. The spectrum (Figure 3.58) Showed that CcO was expressed in the strain PJ L16, which is consistent with the western blot result. 60 TrfA TetA Trb A "" Origin OriT Multiple cloning site CcO subunit 1 gene GGAS EYFP gene CcO subunit “-1" operon EcoRI Figure 3. 4: Plasmid construction of pJL-l6 The gene encoding EYFP (yellow) was linked to the gene of CcO subunit I (blue) through a 4 amino acids linker gene (Gly-Gly-Ala—Ser, white). The GGAS linker was used to help the folding of both EYFP and subunit 1. This subunit 1 —EYFP fusion, together with a fragment containing the subunit II-II operon with a His-tagged subunit II, was then cloned into the expression plasmid pRK415-1 through an EcoRI Site. 61 13 (108 ~ (107 ~ 01x3— 605 nm (105 « l (104 . Absorbance (103 . (102 « (101 - 500 550 600 650 Wavelength (nm) Figure 3. 5: Expression of Subunit I-EYFP CcO fusion protein. A: Western blot result: Lane 1: EYFP protein (Purified from E. coli, used as a positive control), Lane 2: Cell lysates of PJL-C,used as a negative control. Lane 3: Soluble fi'action of JL16 cell lysates. Lane 4: Membrane fraction of PJL16 cell lysates. B: Dithionite-reduced-minus—ferricyanide-oxidized difference spectra of the membrane extract fiom the JL16 strain 62 444nm v 8 — WT CCO 8 0.06 . "g 515nm B I < 0.04 . 0.02 . 0 400. ' 440 ' 480 ' S20 ' 560 ' 600 ' 640 680 Wavelength (nm) Figure 3. 6: Reduced spectra of FPLC purified RchO-EYFP fusion protein. The blue curve is the dithionite reduced spectrum of RSCcO-EYFP. The red curve is the dithionite reduced Spectrum of WT CcO. Besides the 444 nm Soret peak and the 606 nm alpha peak, a new absorbance peak was observed in the spectrum of the RchO-EYFP fusion protein. This absorbance peak is believed to be caused by the EYFP. 63 2.3 Purification of CcO-EYFP fusion protein After the confirmation of expression of CcO-EYFP, the fusion protein complex was purified with a Ni-NTA column followed with a DEAE-SPW anion exchange column. The protein purification procedure is described in Chapter 2. Previous studies Showed that dithionite-reduced CcO has a Soret peak absorbance (444 nm) and a or peak absorbance (606 nm). This absorbance pattern is caused by the heme a and heme a3 of CcO and has been used as the Spectrum fingerprint of CcO (Shapleigh et al. 1992; Zhen et al. 1998). EYFP has a strong absorbance at 515 nm. Thus all those absorbance peaks were expected in the spectrum of CcO-EYFP. The dithionite-reduced spectrum of pu rified CcO-EYFP proved this expectation (Figure 3.6). AS a comparison, the WT CcO only has the Soret peak and the or peak. A fluorescence spectrum was also taken for the purified CcO-EYFP protein. Results showed that this fusion protein has a similar fluorescence pattern as the purified EYF P (Figure 3.7). During the purification, this subunit I-EYFP CcO protein was partially degraded. The running profile of DEAE-SPW column showed that there were 2 different fractions for the CcO-fusion protein (Figure 3.8A). SDS-PAGE was performed to compare the proteins of these two fractions (Figure 3.8B). The results Showed that in the first peak fraction, subunit I-EYF P protein was heavily degraded while in the second peak, the protein is purer than in the first peak. The Spectrum of the degraded form of CcO-EYFP Showed a decrease in the 515 nm, suggesting that the EYFP was cutoff from the subunit I-EYFP full-length protein (Figure 3.8C). Therefore, after this column, the peak 2 fraction was collected and concentrated. 64 30000 t — EYFP excitation scan — EYF P emission scan 25000 - — CcO-EYF P emission scan 200001 15000 - Fluoresence strengh 10000 ' 5000 ' 0 U U U I T T 450 470 490 510 530 550 570 590 610 Wavelength (nm) Figure 3. 7: Fluorescence spectrum of EYFP and CcO-EYFP fusion protein. The left side of this figure is the excitation scan, Shows that the excitation wavelength of EYFP is 515 nm. The right Side showed that the CcO-EYFP and EYFP have the same emission feature. (Bufiter: 50 mM HEPES-KOH, pH 7.4, with 0.1% DDM. Protein concentration: EYFP: 514M, CcO-EYFP: 3.5 uM) 65 Salt Concentration IIIIIIIIIIIIIIIIIIIIIIIIIII iiiiiiiiiiiiiiiiiiiiiiii IIIIIIIII Ll . . A e 'an L.l FPLC fractions 0.06 i 0.05 - —Peak-1 8 004 , —Peak-2 t: ‘3 l 25 0.03 w n i < 0.02 1 l 0.01 . 0 f , r . . . 400 450 500 550 600 650 700 Wavelength (nm) Figure 3. 8: Proteolysis of CcO-EYFP protein 66 1 2 3 4 101kD ' ‘. ...... 4.__ Subunit I-EYFP 56.2kD W 4 - .4 1;“??? ‘_Subunit I 35kD M“ M w ‘— Subunit II 29 kD 4......» .... ' Subunit 111 Figure 3. 8: Proteolysis of CcO-EYFP protein A: DEAE-SPW column running profile B: Dithionite-reduced only spectrum for proteins from peak-1 and peak-2 fraction. The decrease at 515 nm indicated that the EYFP of peak-1 fraction is partially lost. C: SDS-PAGE analysis of proteins from peak-1 and peak-2 fraction. Lane 1: Bio-Rad Prestain Broad Range Protein marker. Lane 2: Purified wild type CcO protein. Lane 3: Purified COO-EYFP fi'om peak-2 fraction. Lane 4: Purified CcO-EYFP from peak-1 fraction. (SDS-PAGE gel concentration: 18%) 67 2.4 Activity assay of RchO-EYFP fusion protein An activity assay was also performed on the purified RchO-EYFP fusion protein. The maximum turnover number (molecular activity) of RchO-EYFP fusion protein and WT CcO was measured polarographically. The results Show that the RchO-EYFP fusion protein has the similar activity as the wild type CcO (Table 3.1). 2.5 Crystallization attempts and results Preliminary crystallization attempts of the ptuified CcO-EYFP protein were performed. Wild type CcO crystallization conditions as described in Chapter 2 have been tried on this fusion protein. No crystal was achieved in either 2 or 4 subunit crystallization conditions. A Hampton crystallization screening kit was also used. Of the 48 different conditions of this kit, some small irregular crystals were formed in one drop. The color of the crystal was dark red, which was Similar to the color of wild type CcO crystals. Four crystals were analyzed at the Advanced Photon Source synchrotron radiation facility, Argonne National Laboratory. However, none of them provided useful diffraction data. This indicated that the quality of the crystals and the crystallization conditions still needed to be improved. 2.6 Use of GFP mutants as indicators of pH in reconstituted vesicle to study the proton pumping mechanism CcO not only reduces O2 to water, but also pumps protons to produce a transmembrane electrochemical potential. The proton pumping and pathways and mechanisms have been studied (Michel 1998; Mills and Ferguson-Miller 1998; Zaslavsky and Gennis 2000). However, the mechanism of coupling of the proton pumping with the oxygen reaction chemistry and the pathway for proton backleak are still unclear. Reconstituted CcO in 68 Proteins Activity (e'/aa3/Sec) WT CcO 11,35 CcO-EYFP 1097 Table 3. 1: Activity of FPLC purified RchO-EYFP fusion protein 69 4H Cytochrome c (4 e-) uncoupler Figure 3. 9: Reconstituted vesicles of RchO-EYFP This is a diagram of Cytochrome Oxidase Vesicles (COVS) and ionophore condition for measuring proton pumping and uncoupling with Photophysics stopped-flow apparatus. Reconstituted cytochrome c oxidase vesicles (COVS) was produced with different concentration of HEPES-KOH, pH 7.4 buffer using the cholate dialysis method. EYFP, which is conjugated onto the subunit I of CcO, was placed inside the vesicle. In the controlled state high membrane potential difference (A‘F) and pH gradient (ApH) is maintained, so that CcO Shows low activity. The membrane potential and proton gradient can be dissipated by adding valinomycin and uncoupler (CCCP) respectively. 70 proteoliposome vesicles (COV) has provided some insights on movement of protons in CcO (Hosler et a1. 1992; Zaslavsky and Gennis 2000; Mills et al. 2002; Mills and F erguson-Miller 2003). The difference between CcO free enzyme and CcO in COVS is that the membrane separates the inside from the outside (Figure 3.9). A membrane potential and a proton gradient is built up during the reaction because the electrons are donated from cytochrome c on the outside, while protons are taken from the inside to support the oxygen chemistry and to be pumped to the exterior of the vesicles. This potential gradient inhibits the activity of CcO. The potassium carrier valinomycin can dissipate the membrane potential by equilibrating potassium across the membrane, while the uncoupler reagents, FCCP (carbonylcyanide-p-trifluoromethoxy-phenylhydrazone) or CCCP (carbonyl cyanide m-chlorophenyl-hydrazone), can eliminate the proton gradient, by equilibrating protons. When both gradients are dissipated, the activity is stimulated giving rise to the “uncontrolled” state of COVS. During this process, by using different pH sensitive dye outside or inside the vesicles, the pH change can be measured and the proton movement pattern can be investigated. However, previous studies in our group showed that if we put a pH sensitive dye, such as phenol red, inside the vesicles, the absorbance signal and properties of the dye appear to be modified when inside the vesicle. Another dye, pyranine, behaves better and is preferred to study inside vesicle pH changes, but its signal is also very weak and hard to quantify due to the small interior capacity of the vesicles (Thesis of Dr. Namjoon Kim, personal communication). The OF P pH sensitive mutant protein can be an alternative candidate for the fluorescent pH probe. In construct pJL16, EYFP is attached to the C-terminus of subunit 1. After reconstitution, the EYFP Should be oriented to be inside 71 the vesicles. The fluorescence emission of EYFP will change according to the internal pH environment. This special characteristic makes it a good fluorescent pH probe to measure the pH change inside the vesicles. However, the size and the hydrophilic characteristics of EYFP might disrupt the vesicle reconstitution. So the first question was, could this EYFP-CcO fusion protein be reconstituted into the vesicles like the wild type enzyme? 2.6.1 Reconstitution of COVS with CcO-EYFP fusion protein Both subunit I-EYFP and wild type CcO reconstituted vesicles were prepared with cholate dialysis method (Hosler et al. 1992) as described in Chapter 2. After the reconstitution, the inside of vesicles contained 75 mM HEPES-KOH buffer and the vesicles were resuspended in 50 uM HEPES-KOH buffer with 45 mM KCl and 44 mM sucrose to stabilized the osmotic pressure. The final concentration of enzyme in COVS is measured by a dithionite-reduced-minuS-oxidized spectrum. 2.6.2 Activity assay and RCR of EYFP-COVS The activity and respiratory control ratio (RCR, the ratio of uncontrolled state rate to the controlled state rate) of EYFP-COVS was measured with a Gilson oxygraph as described in Chapter 2. The purpose of this assay is to compare the activity of RchO-EYFP COVS and WT COVS. The results are Shown in Table 2. Usually the RCR of wild type COVS is in a range of 6~10. From these results we can see that the RchO-EYFP COVS had a similar reaction rate and RCR as the wild type COVS. This means that the EYFP did not affect with the reconstitution of COVS, and that the CcO-EYFP is in its normal orientation with the cytochrome c binding site on the outside of the vesicles. 72 Type of COVS Controlled state Uncontrolled state RCR activity (e'/aa3/sec) activiy(e'/aa3/sec) Measured by oxygen consumption WT CcO 303 1320 6.2 CcO-EYFP s 166 950 5.7 Measured by cytochrome c oxidation (stop flow) WT CcO 201 1470 7.3 COO-EYFP 185 1018 5.5 Table 3. 2: RCR measurement results of RchO-EYFP COV 73 2.6.3 Preliminary fluorescence stop-flow analysis of EYFP-COV The main purpose of reconstituting vesicles with EYFP-CcO was to use the EYFP directly to measure the pH changes inside the vesicle. The stopped-flow method, as described in Chapter 2, was used to measure the cytochrome c oxidation and the proton pumping of EYFP-COVS. RCR and pre-steady state rates of EYFP-COVS were measured with the cytochrome c oxidation assay. Reduced cytochrome c was used as the substrate. Oxidation of cytochrome c will cause the decay of its 550 nm absorbance. COVS were placed in one syringe of stopped-flow instrument and cytochrome c was placed in the other syringe. After mixing the sample with either no ionophores (controlled state) or valinomycin or valinomycin + CCCP (uncontrolled state), scans were collected (1000/S), and from the reduction of cytochrome c (550 nm), the rates were fit using Single exponential fitting to the kinetic traces with Microcal Origin software. The results showed the same pattern and reaction rate as the WT COVS (Figure 3.10). The RCR calculated from these data is 5.5, which is also consistent with the results of oxygen consumption assay (Table 3.2). Proton pumping measurements of EYFP-COVS were also performed. In this experiment the EYFP is used as a pH indicator inside the vesicles. In the measurement, the excitation wavelength was set at 515nm. Fluorescence emission changes were measured at 525nm. The results are Shown in Figure 3.11 .The result showed an increase in the fluorescence level at the beginning of the controlled state. After adding valinomycin into the system, the level of this increase was faster and the level was higher than in the controlled state. This increase indicated the alkalinization due to the proton uptake catalyzed by the CcO. A Similar result was seen with the wildtype CcO reconstituted with the pyranine dye on the inside of the vesicle. 74 0.065 — Controlled state E 0055 — Add Valinomycin 5 0_045 —---- UnControlled state (add CCCP) o . g . '2 0.025 « i o . 8 L < 0.015 4 0.005 4 -0.005 JO 1 2 3 4 5 Time (second) 0.069 — Controlled state g 0059 — Add Valinomycin 8 0.049 .....=.._ UnControlled state (add CCCP) Lo . 8 0.039 c 8 5 0.029 in .Q < 0.019 0.009 -0.001 i——-l——-—|-———-‘" " “ " ' ' " 0 1 2 3 4 5 Time (second) Figure 3. 10: Cytochrome c oxidation by WT COVS and CcO-EYFP COVS. A: WT-COV. B 4: RchO-EYFP COV. For the assay, 2 pM of cyt c2+ was mixed with 0.04 UM of oxidase in the COVS (Inside buffer: 50mM HEPES-KOH, pH7.4 +43 mM KCl). Data scans were collected (1000 scans/S) by Photophysics stopped flow apparatus. 75 — Controlled state ——Add Valinomycin o 2 A o o in E O .2 u. 0 0.5 1 1,5 2 Time (second) 0,02 - —Controlled state —Add Valinomycin 8 0.015- B 5 3 001- 2 . 8 E 0.005 0 . . . , , . 0 1 2 3 4 5 Time (second) Figure 3. 11: Proton pumping by RchO-EYFP COVS A: WT COV, fluorescence dye pyranine was used inside vesicles. B: RchO-EYFP COV, the EYFP in the fusion protein is used as pH fluorescence detector inside vesicles. The Data scans were collected (1000 scans/S) by Photophysics stopped-flow apparatus. For EYFP-COVS, the fluorescence is and detected at 525 nm. For the wild type COVS, the fluorescence is excited at 465 nm detected at 510nm. 76 3. Discussion The CcO-EYFP protein has been expressed in R. sphaeroides and purified. The attached EYFP is soluble and increases the hydrophilic portion of CcO. Preliminary crystallization was also performed on this fusion protein. Although some small crystals were formed, no diffraction was observed with these small crystals. There are several possible reasons: (a) Subunit I-EYFP protein was partially degraded during the purification. After the DEAE SPW purification, most of the truncated protein was separated from the full—length protein. However, SDS-PAGE Showed that there is still some truncated form of subunit I-EYFP in the purified protein (Figure 3.8C). This impurity and non-homogeneity of the enzyme might have undermined the crystallization. An improved the purification method or engineering a more stable fusion protein will be useful in solving this problem. (b) EYFP has likely changed the hydrophilic/hydrophobic character of CcO. The crystallization conditions such as concentration of detergent or precipitant reagents and types of detergent may need to be changed and optimized. (c) Although EYFP may have increased the hydrophilic interaction between protein molecules, the new domain is only attached by one linkage and that is likely to be flexible so that the new domain does not help to make a stable crystal contact. Inserting the EYFP into the CcO by two attachments might help reduce the flexibility Of the construct. My results Showed that the CcO-EYFP reconstituted vesicles had Similar activity and RCR as the wild type CcO vesicles. Another key question is, can EYFP be used as a pH indicator inside the vesicles? To answer this question, fluorescence stopped-flow was performed to investigate the pH sensing ability of the EYFP-COVS. 1n the COVS, the environments of inside and outside of the vesicles are separated. Thus the pH and electrochemical potential 77 changes caused by the proton pumping can be observed independently and quantified. For the CcO-EYFP fusion protein, EYFP could act as a pH sensor to investigate the pH changes inside the vesicles. Proton pumping will cause the pH to increase inside the vesicles, SO we would expect to see an increase in fluorescence signal due to this change. At the beginning of the controlled state, the proton will be pumped out from the inside of the vesicles. For each catalytic cycle, four protons will be used to react with O2 and 4 more protons will be pumped out. However, after the transmembrane potential is built up, the proton pumping is constrained, and the rate of alkalinization inside the vesicle will be Slowed down until a balance is reached. After adding valinomycin, the transmembrane potential of COV is dissipated and more protons will be taken up for substrate and to be pumped out (Figure 3.11). The experiment results are consistent with this postulate. Furthermore, comparing the data of cytochrome c oxidation and proton pumping, we find that the reaction rate and the proton pumping are also co-related (Figure 3.10 and Figure 3.1 1). Since the results obtained with the modified RchO-EYFP were similar to the results obtained with the WT CcO, but with lower Signal to noise, the concept of using the RchO-EYFP as a internal pH monitor appears feasible. However, there is clearly a need for larger vesicles that hold more protein and hence give a stronger signal. Our lab has developed several methods to do this (Dr.Namj oon Thesis; Hiser, personal communication) which will be tested in the future to see how robust this method might be. 4. Summary and Conclusions An RchO-EYFP fusion protein was produced and expressed in Rs strain. The purpose is to increase the hydrophilic portion of Rch0 and form better crystal contacts. The results 78 showed that the fusion protein was fully active. The cytochrome c oxidation rate and proton pumping ability of this fusion protein is very Similar to that of WT CcO, suggesting that the fusion of EYFP did not affect the normal function of the enzyme. The reconstitution of the fusion protein into liposome vesicles was successful and fluorescence changes could be observed according to the pH changes inside the vesicles. The fluorescent signal was still weak for accurate proton pumping measurement, better conditions still need to be developed, such as making of larger vesicles, lower internal buffering capacity etc. Extensive crystallization screening on this fusion protein was also performed, however, no evidence that the fusion helped crystallization. This result could due to the partial proteolysis of the fusion protein, suggesting a two-Site attachment would be worth trying in the future, given the close positioning of the N and C terminals of EYFP. 79 CHAPTER 4. STUDY OF THE SUBUNIT I C-TERMINUS MUTANT FORMS 1. Introduction Following the first successful crystallization of the four subunit CcO from Rhodobacter sphaeroides(Svensson-Ek et al. 2002), neither the Swedish group nor our group were able to reproduce the level of resolution initially achieved (2.3-2.8 A, anisotropic, in only one crystal) in Spite of several years of effort in both groups. Our success in producing a new, reproducible, high resolution (2.0 A, isotropic, Qin et al., 2006) crystal form that contained the two core catalytic subunits did not obviate the need to continue our efforts to obtain a better four subunit structure. Previous studies have Shown that removal of the flexible portions of certain proteins can help the protein crystallize or improve the crystal quality to get better diffraction data The last 16 amino acids of the C-terminus of RchO subunit I are not Observed in the 2-Subunit crystal structure, indicating this end may be flexible in nature. In the 4-subunit crystal of Rch0, 6 amino acids are missing (Figure 4.1). This apparently flexible end might interfere with the crystal contact formation as mentioned in Chapter 3. A possible solution is to use molecular engineering to remove the flexible portion of subunit 1, to create a new C-terminus with new crystal contacts, and improve crystal quality. This region is not highly conserved, adding to the likelihood that its removal may not disturb the function of Rch0. 80 l 1M56 subunit 1 1 '2" .,\ PL. _._.._ A.- l '1 'A . ._' :- Q I . . | . ' ‘ . u“ v1 3 u c u‘ C‘ 9! J 3.1" ‘9.- ‘4 I RRYIDYPEAFATWNFVSSLGAFLSFASFLFFLGVIFYTLTRGARVTANNYWNEHADTLEW l 56° ,_ 1M56 . ' ._ 4subunit CcO crystal TLTSPPPEHTFEQLPKREDWERAPAH I 1M56 subunitl l 9 ‘ , .' '.._ n RRYIDYPEAFATWNFVSSLGAFLSFASFLFFLGVIFYTLTRGARVTANNYWNEHADTLEW l ...—“550 _ - 2GSM: Zsubunit CcO crystal TLTSPPPEHTFEQLPKREDWERAPAH Figure 4. l: C-terminus comparision of 4-subunit RchO crystal and 2-subunit CcO crystal. These results are from the crystal structures OfRchO 1M56 and 2GSM. In structure 1M56 (4 subunit crystal of RchO), last 6 amino acids of C-terminus of subunit I were not defined (blue letters). In crystal structure 2GSM (2 subunit crystal of RchO), last 16 amino acids of subunit I C-terminus were defined (blue letters, and the F551 is always poorly defined). These residues could be very flexible in the structure. 81 Anther potential problem for crystallography is subunit III of RchO, a highly hydrophobic transmembrane subunit with some tendency to be lost during purification. The 4-subunit crystal structure of RchO Shows that it has multiple lipids bound (Svensson-Ek et al. 2002) and could be very flexible in the structure. It is possible that the instability and partial loss of subunit III is another factor that affects the crystal quality of RchO. Crystallization results of 4-Subunit and 2-subunit crystals ofRchO are consistent with this hypothesis. The 2-subunit crystal of RchO, which does not contain subunit III or subunit IV, has consistently higher resolution (routinely 2.0~2.5 A) compared to the 4-Subunit crystal of RchO (routinely 2.9~3.5 A). To stabilize subunit III in the 4-subunit crystal could be another way of improving the crystal diffraction quality. The known crystal structures of RchO Show that the C-terminus of subunit I is very close to the N-terminus of subunit 111 (Figure 4.2), suggesting a possible interaction between these two subunits at this region. Through molecular engineering, we can link the C-terminus of the subunit I gene to the N-terminus of the subunit III gene, to stabilize subunit III in the whole structure, preventing its loss during purification and thus improving the crystal quality of the 4-Subunit crystal of RchO. In this chapter, two mutants of RchO containing short versions of subunit I are described (PJL33 and PJ L49) and characterized to investigate the effects of C-terminal deletions on the crystallization. A single mutant at residue E552 was also created to test the possibility that it might be responsible for observed loss of pumping in one of the shortened versions. A subunit I-subunit III fusion mutant, PJL63, was also created to study the effects of subunit III on the crystallization. 82 a .5— ~__.;_ '0'" - \‘lz‘t ... . ..f, we hi3 ‘33:” Serf? «««« Figure 4. 2: Possible interaction between subunit I and subunit III of Rch0. The subunit III is shown in purple color. The subunit I is shown in red color. The yellow colored loop end region is the C-terminus of subunit 1, from residue number F551 to W560. This region could not be resolved in the 2 subunit RchO due to its flexibility in the structure. The N-terminus of subunit III is very close to this region, providing the possible interactions in subunit I C-terminus. 83 2. Methods and Results 2.1 Construction of the short subunit I CcO mutants PJL33 and PJL49 The construction of short subunit 1 RchO mutants F] L33 and PJL49 is shown in Figures 4.3 and 4.4. In the plasmid construct pJL-33, the last 16 amino acids of the subunit 1 C-terminus were deleted, pJL-33 contains a his-tagged subunit II and wild-type subunit III operon (Figure 4.3) for the purification. In the plasmid construct pJL-49, the codon of E561 was mutated to a stop codon so it does not contain the last 6 residues of subunit I after translation (Figure 4.4). The plasmid constructs were then conjugated into the R.sph D 1 D4 strain by using the method described in Chapter 2 to produce two strains (PJ L33 and PJL49) that express Short subunit 1 RchO. 2.2 Protein expression and purification of PJL33 and PJL49 The proteins of PJ L33 and PJL49 were expressed and purified as described in Chapter 2. The reduced-minus-oxidized spectra of the membranes showed normal spectra for both PJL33 and PJL49 compared to the wild type membrane Spectrum (Figure 4.5), suggesting the expression of PJL33 and PJL49 was successful. The expression level of both F] L33 and PJL49 are quite high judging from the ratio of the 550 nm peak to the 605 nm peak. The proteins of PJL33 and PJL49 were then purified with the FPLC-NiNTA column. A further purification step of a DEAE-5pw column was also performed to facilitate the RsCOV reconstitution. 84 TrfA TetA . OriT TetR‘ Multiple cloning site CcO subunit IA551—566 _‘ CcO subunit Il-histag subunit 111 Pstl Pstl Figure 4. 3: Construction of pJL-33 expression plasmid The subunit I gene missing the last 16 amino acids was cloned into the pRK415-1 plasmid at the Pst I site together with a fragment containing subunit II-III operon. His-tagged subunit II was in the operon and can be used to purify the enzyme with a Ni-NTA column. 85 Tl'fA .5“? TetA OriT Multiple cloning site C60 subunit 1 5561* CcO subunit [1 histag-III operon "— Pstl Pstl Figure 4. 4: Construction of pJL-49 expression plasmid The subunit I E561 codon was mutated into a stop codon, so the expressed protein will be 6 residues less in C-terminus of subunit 1. 86 0.048 : PJL33 0.038 ‘ 0.028 ‘ 0.018 r 0.008 : -0.002 ‘ -0.012 - -0.022 ‘ “0.032 I r I T I I Y I I l I 500 520 540 560 580 600 620 640 660 680 700 0.04 2 003 ~ M 0.02 — 0.01 ‘ PJL49 —0.01 ‘ -0.02 ‘ -0.03 - 004 . . j 1 500 550 600 650 700 Figure 4. 5: Reduced —minus-oxidized spectrum of the membrane fraction of PJL33 and PJL49 CcO The at peak (605 nm) from PJL33 and PJL49 is relatively high compared to the 550 nm bcl complex peak, suggesting a high expression level of CcO in the membrane. 87 2.3 SDS-PAGE of purified PJL33 and PJL49 CcO The SDS-PAGE was performed to verify the result of the purified PJL33 and PJL49 CcO (Figure 4.6). The result Showed that in both PJL33 and PJL49 CcOs, the subunits I are Shorter than the wild type RchO subunit 1, and the Size of subunit I from PJL49 is slightly larger than the subunit I from PJL33, indicating the mutations were successful constructed in these two strains. The SDS-PAGE also showed that in purified PJL33, the amount Of subunit III was much less than the amount of subunit 111 from wild type CcO. Considering the possible interaction between the subunit I C-terminus and the subunit III N-terminus, this reduced amount of subunit 111 could be the result of the loss of certain residues among the 16 deleted from subunit I in P1 L33, thus the loss of the interaction. However, PJ L49 contained a similar amount of subunit III as wild type RchO, suggesting the effects in P1 L33 could be caused by the deletion of the 551 to 561 region of subunit 1. 2.4 UV-vis spectra of purified PJL33 and PJL49 CcO The reduced Spectra of P1 L33 and PJ L49 CcO were measured as described in Chapter 3. The results are shown in Figure 4.7. The reduced Spectra of PJL33 and PJL49 purified proteins were very similar to that of WT RchO, with the 444nm Soret peak and the 606nm a peak, suggesting that the deletion did not affect the heme centers of RchO. 2.5 Activity of PJ L33 and PJL49 The steady state activities of purified PJL33 and PJL49 proteins were measured over a range of pH conditions. The method used was described in Chapter 2. The results are 88 60kD Subunrt I 30kD g}, >..— Subunit 11 SubunitIII 26kD ' Subunit IV ~33. bank‘? 6kD * ’ Figure 4. 6: SDS-PAGE of FPLC purified PJL33 and PJL49 protein Lane 1: NEB protein ladder 2: W.T CcO (Full length subunit I) 3: PJL49 ( 6 residues deletion subunit 1) 4: PJL33 ( 16 residues deletion subunit 1) 18 % urea SDS-PAGE 89 0.457 1 444 nm 0.4 0 35 — PJ L33 oxidized ' . — PJL33 reduced 0.3“: 3 0.25~ 8 E 0.2 8 ii 015‘ 0.1 ~ 0.05- o . , . r . . 400 450 500 550 600 650 700 Wavelength (nm) 0.14 . 444nm 0.12 4 — PJL49 oxidized d.) O .5 H B <‘. 400 450 500 550 m0 650 Wavelength (nm) Figure 4.7 : Spectrum of FPLC purified PJL33 and PJL49 CcO The reduced spectrum of purified PJL33 and PJL49 CcO showed the normal soret peak (444 nm) and on peak (606 nm), suggesting normal redox centers in both proteins. 90 shown in Figure 4.8. Compared to the wild type Rch0, the mutant PJL33 had much lower activity, especially at the high pH region. In the low pH region,the activity of PJL33 was about 60% of wild type enzyme activity. In the high. pH region, the activity of PJL33 was only 30% of wild type. Also, as the pH increased, suicide inactivation was observed during the reaction (Figure 4.9) evidenced by the slowing of the turnover rate during the assay. Unlike the l6-residue deletion construct PJL33, the overall activity of PJL49 is at the same level as the wild type RchO. No suicide inactivation was observed in the enzyme reaction of PJL49. 2.6 Proton pumping assay results of PJL33 To further study the characteristics of PJL33, the pre-steady state activity and proton pumping ability were also investigated. The stop flow results are Shown in Figure 4.10 and Figure 4.11. In the cytochrome c oxidation assay, the PJL33 Showed a slower reaction rate when a membrane potential was present across the COV membrane (controlled state) as Shown in Figure 4.10. When valinomycin and FCCP were added to eliminate the transmembrane potential, the reaction rate increased, Similar to wild type enzyme behavior, showing the phenomenon of respiratory control. The pH change outside the COVS was measured with phenol red, which indicates proton concentration changes on the outside of the membrane. From the experimental results, the proton pumping ability of PJL33 COVS is much lower than that of wild type COVS (Figure 4.11). The loss of activity could be the result of losing key residues in the 551-566 region of subunit 1. There are several conserved residues in this region, such as E548 and E552. However, the result of the mutation or deletion of these residues is still unknown. 91 1600 1400 - 0 ’ W-T ' OPJL49 1200 1. g 1000 E 800 . E 600 - .2. 34’ 400 - 200 - 0 I I I l I I 6 6.5 7 7.5 8 8.5 9 pH Figure 4. 8: Activity curve of PJL33 and PJL49 compared to WT CcO The maximum turnover number (molecular activity) of CcO was measured polarographically by using a Gilson model 5/6 H oxygraph at 25 °C in a reaction medium containing a series of buffer with different pH, in the presence of 0.05 % DDM, 2.8 mM ascorbate, 0.55 mM TMPD (tetramethyl phenylene diarnine) and 30 pM of horse cytochrome c. 92 100.0% 90184 80.0% : ...pJL33 7tiorti ' ""'VVJ: 1r1011~ 50.0% * 40.0% f 30.0% ' Oxygen concentration 20.0% , 10.0% 1 0.0% 1— .4 l - l . . o 100 200 300 400 500 600 700 Time Figure 4. 9: Suicide inactivation of PJL33 CcO The blue curve is the oxygen consumption by WT CcO in the Gilson model 5/6 H oxygraph reaction. The black curve is the the PJL33 CcO reaction curve. The dotted line is the initial reaction rate of the enzymes. It shows that, as reaction proceeds, the activity of PJL33 CcO is quickly reduced. This iS always observed in the subunit III less CcO. 93 PJL33 COV Absorbance(549 nm) Time (seconds) 0.035 0.030 0.025 0.020- 0.0155 0.0103 0.005; 0.0001 00053 Absorbance 549nm Time (seconds) Figure 4. 10: Cytochrome c oxidation of PJL33 COVS measured by stopfiow. The curves were created by measuring the absorbance change of cytochrome c at 549 nm during the reaction.The black curve is the controlled state. The blue curve is the reaction after valinomycin was added, and the red curve is the reaction after CCCP was added, which is the uncontrolled state. 94 Absorbance (557 nm) .002. PJL33 COV -0.03 . . . . . 0.0 0.5 1.0 1.5 2.0 Time (seconds) .0 .o .0 O O O —3 N 00 L L l n 1 0.00 «a -0.01 - -0.02 - Absorbance (557 nm) '003 ' I fl I . I r 1 0.0 0.5 1.0 1.5 2.0 Time (seconds) Figure 4. 11 Proton pumping assay results of PJL33 COV and WT COV. The black line is the controlled state. The blue curve is measured by the absorbance changes after valinomycin was added. Less change in PJL33 COVS blue curve suggest that the proton pumping was impaired. The red curve is the uncontrolled state, measured by adding FCCP. 95 2.7 Construction of subunit I C-terminus single mutant E552A To further investigate the reason for the loss of activity and proton pumping in PJL33, a Single mutant E552A was also created. The mutation was first made on the plasmid PJS3-SH using the Qiagen QuikChange kit. The PCR primers that were used for the mutagenesis are Shown in Figure 4.12. The E552 of the subunit I was mutated into an alanine. After the mutation, a fragment containing subunit II-III operon gene was inserted into the Pst I site on PJS3-SH to form plasmid pJL-44. The DNA fragment containing subunit I with the E552A mutation and the subunit ll-III operon was then ligated into pRK415-1 to form the expression plasmid pJL-45. pJL-45 was then conjugated into the R. sph strain A123 as described in Chapter 2. 2.8 Expression and purification of E552A E552A protein was expressed and purified as described in Chapter 2. The membrane fraction reduced-minus-oxidized Spectrum (Figure 4.13) Showed the normal spectrum like WT Rch0. The oxidized and reduced Spectra of purified E552A protein are shown in Figure 4.14. The reduced Spectrum showed a normal Soret peak at 444 nm and alpha peak at 606nm. These results suggested that the heme centers in the enzyme were not disrupted by the mutation of E552 to A552. 2.9 Activity assay of E552A The steady state activity of purified E552A protein was measured using the same method as used in Chapter 2. The result (Table 4.1) showed E552A is fully active at pH 6.5 and pH 8.6. No suicide inactivation was observed during the E5 52A reaction. 96 Upper primer: CCGGAGCATACGTTCGCGCAGCTTCCCAAGCGG Lower primer: CCGCTTGGGAAGCTGCGCGAACGTATGCTCCGG Figure 4. 12: Primers used in the construct of PJL34: E552A mutant. The underlined GCG and CGC Show the mutation fiom gluatrnic acid codon to alanine codon. 97 0.015 ' 605nm 0.01 : § 0.005 ~ (5 19 O U) 3 0 ‘ -0.005 * '0.01 ' I l I l 500 550 600 650 700 nm Figure 4. 13: Reduced minus oxidized spectra of PJL45 membrane fraction. The normal 605 nm peak and high 605 nm/550 nm ratio suggest that PJL45 was highly expressed in the membrane. 98 0'09 l 444 nm 0.08 : 0 07 —552A oxidized spectra 0.06 . “-55% reduced spectra 8 g 0.05 ‘ g 0.04 < 0.03 * 606 0.02 - ”m 0.01 ~ - 2 A 0 I T r r T— r 400 450 500 550 600 650 700 nm Figure 4. 14: Spectra of purified PJL45 CcO. Purified PJL45 CcO (E552A) shows the normal 444 nm Soret peak and 606 nm or peak, indicating the redox centers in PJL45 CcO were not affected by the E552A mutation. 99 pH 6.5 pH 8.6 WT CcO 1243 (i200) 960 (2210) PJL45 (E552A) 1200 (3:280) 848 ($250) Table 4. 1: Activity of PJL45 CcOat pH 6.5 and pH 8.6 Acitivty was measured by oxygen consumption with purified PJL45 protein. Activity Unit: (e'/sec/aa3) 100 Absorbance (557nm) O 0.5 1.0 1.5 2.0 Time (seconds) 1 0.02 - E 0.01 - (\ V3 3 ,. 8 0.00 i. 8 ..D ‘8 g -0.001 - PJL45 COV -0.002 ‘ 0 0.5 1.0 1.5 2-0 Time (seconds) Figure 4. 15: Proton pumping assay of PJL45 COV. Black: controlled state. Blue: Valinomycin added. Red: FCCP added, uncontrolled state. PJL45 COV (lower chart) showed less proton pumping compared to WT COV (upper chart). 101 E552A was also reconstituted into vesicles using the methods described in Chapter 2. The proton pumping ability of E552A was measured by stop-flow and compared with WT Rch0. The results are shown in Figure 4.15. From the results we can see that the proton pumping ability of E552A COVS was Slightly impaired compared to that of WT COVS. However, more experiments are still needed to comfirm this result Since the instability of the proton pumping measurement in COVS. 2.10 Crystallization of PJL33 and PJL49 Both 4-subunit and 2-subunit crystallization conditions were tried on the purified PJL33 and PJL49 proteins. There were no crystals of PJL33 and PJL49 formed under the 4-subunit crystallization conditions. However, purified PJL33 could be crystallized with 2 subunit crystallization conditions. Six 111 Of enzyme solution containing 10 mM Tris, pH 8.0, 50 mM NaCl, 0.20% decyl maltoside and 120 11M CcO was mixed with 3 uL of reservoir solution containing 100 mM MES, pH 6.3, 27% PEG-400 and 3 pl Of crystallization additive solution containing 5% heptanetriol, 32 mM MgCl2, 1.3 mM CdCl2 and 0.026% dodecyl maltoside. Two subunit crystals of PJL33 were observed after 3 days growing under 4°C incubation. After two weeks, the crystals of PJL33 were collected and examined at Argonne National Lab. The best resolution of the crystals is 2.1 A (Table 4.2). Two-subunit crystals of PJL49 were also obtained under the sitting drop condition with Slightly different conditions: well solutions containing 100 mM MES, pH 6.3, 27-29% PEG-400. The drop also contained 1.3% heptanetriol, 8 mM MgCl2, 0.4 mM CdCl2 and 0.06% dodecyl maltoside. The best resolution of the crystal of PJL49 is 2.5 A (Table 4.2) 102 PJL33 I PJL49 A. Unit cell parameters Space group: p212121 Space group: [321212] Cell dimension: 01=123.005 0:130000 y=177.539 a=124.905 13=131-477 y =176.601 x=y=z=90.00 x=y=z=90.00 Molecules per A.U. : 2 Molecules per A.U. : 2 B. Data collection: Resolution range: 50.0-2.10 A Resolution range: 50.0-2.50 A Completeness: 99% Completeness: 95% Number of unique reflections: 165884 Number of unique reflections: 117531 Redundancy: 7.3 Redundancy: 5.7 Rmerge=7 Rmerge=4 C. Structure refinement R factor/R flee: 238/25.5 (%) R factor/R free: 26/28.3 (%) RMSD bond length: 0.008A RMSD bond length: 0.022A RMSD bond agle: 1.34 RMSD bond angle: 1.97 B-Factor: 45.4 B-Factor: 52.3 Table 4. 2: Crystal parameters of PJL33 and PJL45 CcO 103 2. 11 Crystal structure of PJL33 Although the crystal of PJL33 has a similar space group (p212121) as the wild type RchO 2-subunit crystals, the cell dimensions of the crystals are Slightly different from that of wild type crystals (Table 4.2). This might be caused by the deletion which affected the crystal packing. Thus molecular replacement, instead of rigid body refinement, was performed to get the right phase information of this crystal. After several refinements, we can see that the overall structure of this mutant is not much different from wild type enzyme. The positions of important redox centers remain unchanged (Figure 4.16). The major structural changes in the PJL33 were found in the T550 region (Figure 4.17). From the crystal structure, we can see that in WT, there are 2 waters bound between T550, H26 and K27, while the distance between these 2 waters is very close (2 A), indicating one of them could be a metal ion such as Mg or Cd. (More experiments are still needed for this hypothesis). In PJL33, the loss of the last 16 residues on submit I make the T550 the new C-terminus. The waters between T550 and H26 are gone. The T5 50 sidechain has now folded in closer to the H26, forming a hydrogen bond between them. This region is very close to the entrance of the D-pathway at residue D132. (Figure 4.17). The changes in this region, including the loss of the waters (or metal) and direct interaction between T550 and H26, could change the pKa of residues in the environment of the entrance of D-pathway which could explain the altered pH dependence of activity and reduced proton pumping of this mutant. Comparing this structure with wild type Rch0 and other mutant structures will help us to further understand the regulation of the D-channel in enzyme function and the proton pmnping mechanism. 104 .o.‘.‘ "I I I Q 1 \‘ ‘ l \ ‘\ . ......x .... . i . 03.0.3.9 f... c ‘48.-.. r- \ .3. v.r . ca .L’rNO-fi. 0". «.0. 304%. .00... 4 .r 69901... humus“: .90. .... . ...... {h 3 .91 1.4 d it???» 1.1/two.- fl \'bzl. \ofihvstQui m 1‘1 .o 8.1“ c.hi¢.".wi 0" ...0 “flute...” n... . v 0 B \. .esu. . .17!) r... r 3...... . a». .I. I as. new! 6 ’4 ~09“: . m... - i inns . . .. u 4.0 Q 4914...”... ‘ I.“ . 'MQW.‘ 9‘15! L“.~ .01..” I.\.\.‘I\1 . . ..r.'~¥ P J - 8 1‘ A ..IsVQWK - . T“.‘ .u . O”, .o I 8 . . . . .. . (I ..I o I a .1... . .. qsaxwfi We»... “3?an . H . n .... \4 (Q... ...».oC.oun..~\. . _ it”. I .. , . 4o ..0 / ‘1‘ a»..\ .. r/ a“ 1‘ t .0 4’ v . H r “..' ‘\ . . "‘ r .. v -’ ‘\‘. A O ‘4’ .I. . ,.0 . v .‘\ A ‘ i \‘D a i . c.4........c..~eu.,. .o._...\\un»\ltil4.7b c \hDOV-V. Io/ .f . I‘ v. . I I. . I .W 01‘ . Q . «wetnnfifi... hxoo radish 6 .51.? in ..,.._.., .. ”A 1 . ‘0. ‘44 o‘ .? D Q 1'7 '4‘ . \‘d"\»...‘ 3 n . ' \S . . k ~w. ~9H”§.\§~U.nlé¥:.h)hu\u. h. a . ... 1.0.1.5997: 341114“ \I‘. r. . .0 MI 1. l V e i3 _ I 4.. .... m . .-f... ‘4444. N... :45 box . . s . ’Qhfildlr H "I s. 88...... an? x a . 1.90 ms». % PDQ. ‘r.h\'u\.§~. .s ‘4 Chablr/ 4.. h. . pl)... 3...“...OM ”any... which... “.... an», we». WWI... ... '53an 9.9. ‘ . s .. ....V'V ‘Jvflovmwmv .‘w'; 7“?“ . -.....sO/Ile». x»)? 0.6... $.31 .... 1... ......aa 4&5on -. .s. x} ...0! ...“...v. - ....uw... 1 Swift. r». J. an. ..‘\.s.. .., , ”5' .. . [.0 . . . um,“ .WWWHI 676.115- r .4.... . wv."‘. \ i ..9 I .' «It ‘ SAIL.‘ ‘1 . i Q .‘ 4 I i II), .33.. ..K . a .4.... Eve . . :xhfi .VO.‘\N;'\. ‘.0.. ‘9‘“. . .I- .tflflhdflg with WT 0.0 the WT CcO structure 105 lor. The yellow structure is tro gen atoms and oxygen atoms respectively) Figure 4. 16 Structure alignment of the redox centers of PJL33 PJL33 structre are shown in green co (2GSM). (Blue and red are the ni Figure 4. 17: Structure comparsion of PJL33 and WT CcO at C-terminus region The waters are rendered in red Spheres. The 2fo-fc map (blue mesh) showed that the mutation in PJL33 makes the T550 became the new C-terminus of subunit IH, with the loss of the waters in between H26 and T550. A new hydrogen bond interaction was formed between H26 and T55 0 directly. 106 2.12 Crystal structure of PJL49 CcO The 2-subunit crystals of P] L49 CcO were examined at LS-CAT at Argonne National Lab and the best of them could diffract to 2.50A (Table 4.1). The overall structure of PJL49 is very similar to the WT CcO structure. It has the same space group (13212121), Similar unit cell dimensions and crystal contacts. After a few rounds of refinement, we can see that the main metal centers of this mutant, heme a and the heme a3-CuB site, are not altered. Unlike the T550 region of PJL33 where there is a Significant structural change and 2 water molecules that bonded to T550 and H26 were missing, in PJL49 the T550 is in same conformation as wild type CcO, as are the 2 water molecules /metal (Figure 4.18). These structural differences could account for the enzyme activity difference between PJ L49 and PJL33.A11 these results Showed that P] L49, having lost the last 6 residues from subunit 1, is almost the same as wild type CcO in the aspects of enzyme activity and structure. This cut off site does not affect the normal function of wild type C00 and likely provides a more stabilized C-terminus. It can therefore be used for a new construct of CcO with a gene fusion to potentially get better crystal contacts and higher resolution. 2.13 Construction and Preliminary study'of the subunit I-III fusion protein : PJL63 An expression plasmid, pJL-63 was constructed to express the subunit I C-terminus subunit lII-N-terminus fusion mutant. The C-terminus of subunit 1 (ending in residue 561) and the N-terminus of subunit III gene (starting from residue 6) were linked together by PCR. A subunit H gene with his-tag was also cloned into the pJL-63 plasmid construct. 107 WT PJL49 Figure 4. 18: Structral comparison of the C-terminus region of WT CcO and PJL49 The water is rendered in red spheres. In the PJL49 figure the 2fo-fc density map shows the mutation did not afi'ect the c-terminus H26-water-T550 interaction. 108 0.045 - 0.04 . M 0.035 4 (103 ‘ Absorbance .0 8 O N N 01 0.015 < 0.01 J 0005 : 500 550 600 650 700 nm Figure 4. l9: Reduced-minus-oxidized spectra of PJL63 membrane fraction The spectra Show that the expression level of PJL63 is fairly good. And the enzyme showed a normal spectra (605 nm or peak) while inside the membrane. 109 0.25 - 443 nm 0.2 1 —PJL63 oxidized —PJL63 reduced _ . § 0.15 1 as e 8 E 2 0.1 603.8 um I 0.05 1 0 I I I r I I 400 450 500 550 600 650 700 nm Figure 4. 20: Reduced and oxidized spectra of FPLC purified PJL63 Both soret peak and a peak are shifted in the purified PJL63 CcO, suggesting the redox center of this enzyme could be changed. 110 The plasmid was then conjugated into R.sph strain A123 as described in Chapter 2 for expression. The PJL63 protein was expressed and pm'ified as described in Chapter 2. Before the purification, the reduced—minus-oxidized spectrum of the membrane fraction of PJ L63 was taken and it looked the same as wild type RchO (Figure 4.20). However, the reduced spectrum of the Ni-NTA FPLC purified PJL63 protein (Figure 4.21) showed a lower and Shifted Soret peak (443nm) and shifted peak (603.8 nm), suggesting a possible loss of heme a3 and disruption in the heme a region. The steady state activities of purified PJL63 were measured at pH7.4 as described in Chapter 3. Compared to the wild type Rch0, the mutant PJL63 had a lower activity (860 e'/sec/aa3 vs 1090 e'/sec/aa3) and a tendency toward suicide inactivation — inactivation during turnover. These results and the altered spectrum of PJL63 suggested that this fusion protein might not be stable in structure. The firsion could have caused a conformational change in subunit ‘1 and subunit III and thus affected the heme region environment and the enzyme activity. 3. Discussion The aim of the studies described in this Chapter was to examine several possibilities of improving the quality of RchO crystals by removing flexible ends and creating gene fusions so as to improve the stability and molecular homogeneity of the protein. Of particular interest was to facilitate production of four subunit crystals at higher resolution than previously achieved. The results were disappointing in this regard, but did reveal some interesting functional information about the C-terminal region of subunit 1. 111 Although the C-terminus of subunit 1 is not highly conserved and Shows evidence of flexibility, there are some residues in that region that appear to be important to the enzyme fimction. This region is near the D132 residue, at the entrance to the D-channel in CcO. A report on the bovine heart CcO structure Showed that a histidine residue at the subunit I C-terminus may be involved in stabilizing the water network in this region and could serve an important role for proton uptake (Muramoto et al. 2007) . According to the sequence alignment, E552 in the deletion region is also a conserved residue that could play a role in proton uptake. However, steady state activity assay results Show that the E552A RchO mutant has similar activity to wild type CcO, suggesting that the loss of E552 itself may not be the cause of this loss of activity in PJL33. Still, it does not exclude the possibility of other residues in the deletion region being very important to the enzyme function, or the activity loss being caused by multiple changes of several residues together. The 4 subunit crystal structure of RchO suggests that the N-terminus of subunit III interacts with subunit I C-terminus to stabilize subunit III on the whole enzyme. Studies on the subunit III deletion enzyme Show that one role of subunit III is to stabilize and protect the enzyme from suicide inactivation during reaction (Bratton et al. 1999). The mechanism of suicide inactivation is apparently not due to the production of reactive oxygen species since adding catalase and superoxide dismutase had no effect on the rate of inactivation (Bratton et al. 1999). SDS-PAGE of the PJL33 protein suggested that after Ni-NTA and DEAE-5pw FPLC columns, the amount of subunit III was reduced compared to the Wild type enzyme. Without subunit 111, although CcO can maintain a high reaction rate at the beginning, it undergoes suicide inhibition and loss of activity. Suicide inactivation in subunit III less 112 enzyme is ultimately associated with the loss of CuB at the heme a3-CuB binuclear center as measured by EPR Spectroscopy and metal analysis, possibly due to the dissociation of one or more of CuB’S ligands (Hosler 2004). Judging from the crystal structure of PJL33, however, the redox center is not affected, suggesting that this loss of subunit III in PJL33 is not servere. Research results also Showed that the rate of proton uptake through the D pathway, as well as the proton exit/back flow pathway, was slowed down in the absence of subunit III (Gilderson et al. 2003; Mills et al. 2003). On the other hand, arachidonic acid, which enhances the D pathway proton uptake, slows down the rate of inactivation of subunit III-less CcO. The role of subunit III could be to maintain a conformation of C00 that facilitates proton flow to the active site through the D pathway and proton backflow through the exit path to the active site (Mills and Hosler 2005). Although we can get high resolution structure of RchO from the two subunit crystal, this suicide inactivation effect caused by the loss of subunit 111 showed the importantce of keeping the subunit III in the whole enzyme. Our ultimate goal is still to get high resolution structures of the whole enzyme. In an effort to stabilize the subunit III and subunit 1 together, the subunit I C-terminus-subunit III N-terminus fusion mutant (PJL63) was made. However, initial studies on the properties of this fusion protein showed that the activity of PJ L63 is affected. The structure of this enzyme could be disrupted, Shown from the reduced spectra. The choice of the fusion point could be very important. More works are still needed to make the subunit I-III fusion protein a better candidate for the crystallization. 113 4. Conclusion and Summary (1) Two mutants of RchO with shortened versions of subunit I (PJL33 with loss of 16 residues at C-terminus, and PJL49 with loss of 6 residues at C-terminus) were created, expressed to good levels and characterized. The results Showed that PJL33 has possibly lost some amount of subunit III and has lower activity and proton pumping compared to wild type RchO. Another version of RchO with Six residues removed from subunit I , P149, Shows normal activity and spectra compared to wild type RchO, indicating that the last 6 residues of subunit 1 do not affect the activity of RchO. Thus, Site 561 was used as a fusion point in future research. (2) Crystal structures of PJL33 and PJ L49 were obtained in the 2 subunit form, but no four subunit crystals were found. Both mutant crystals showed a similar overall structure as the crystal structure of wild type RchO, suggesting the deletion did not change the overall conformation of RchO. In PJL33 structure, the conformation of C-terminus was changed because of the deletion of the last 16 residues. T550 became the new C-terminus of PJL33 and 2 water-molecules that were bonded to it were lost. These changes could be the reason for the loss of activity and proton pumping of PJL33. (3) A subunit I-III fusion mutant PJL63 was created. In this mutant, the C-terminus of subunit I and N-terminus of subunit III were fused together by molecule engineering using the subunit III gene (with the deletion of first 6 amino acid at N-terminus) and the 6-amino-acid truncated subunit I. Although the enzyme was produced in good amounts and Showed a normal spectrum in the membrane, the reduced Spectrum of purified enzyme Showed disruption in the heme a and heme a3 regions. The activity of the purified enzyme 114 was only 80% of wild type RchO. These results indicated that this fusion could disrupt the subunit I and subunit III interaction, thus causing the activity change. 115 CHAPTER 5. CRYSTAL STRUCTURES OF PROTON UPTAKE PATHWAY MUTAN TS OF RSC C O (a paper based on the studies reported in this Chapter is in press, Proc.Natl.Acad.Sci. See Appendix) 1. Introduction Two proton uptake pathways, the D-pathway and the K-pathway, were found in Rch0 by structural studies and site-directed mutagenesis (Fetter et al. 1995; Hosler et al. 1996; Wikstrom et al. 2000; Pawate et al. 2002). Research also showed that these two pathways are highly conserved among mammalian and bacterial CcOs(Ferguson-Miller and Babcock 1996). However, in the bovine CcO structure an additional pathway, the H-path, has been identified (Tsukihara et al. 1996; Yoshikawa et al. 1998). which appears not to be conserved in the bacterial CcO (Lee et al. 2000). The findings in the bovine CcO have led to the proposal of an alternative mechanism of proton pumping and questions regarding whether the process of proton transfer and energy transduction is conserved. This critical issue emphasizes the importance of defining the detailed Structure of proton pathways. In RchO, the D-pathway starts from a conserved aspartic acid D132 (D124 in Pd CcO and D91 in bovine CcO) in subunit 1 and ends near the glutamic acid E286, which is close to the binuclear heme a3-Cu3 center (Figure 5.1). Removal of the carboxyl at position 132 strongly inhibits enzyme activity (2% of wildtype Rch0) and proton pumping (Fetter et al. 1995). Several hydrophilic residues in the D-pathway are crucial for the function of proton uptake as evidenced by the effects of their mutation on enzyme activity 116 / J ,/ l l a \e \ I ‘ ‘< \ Heme a t ’ T c J ( -___, (TX 82 / ‘/ Y33 / \ ‘7' M A \ ‘ ‘ ‘ I, ‘ . I \ ‘ .. ' J \ ’ ’ ‘ ~jf l \ N139 ‘ V ' 5 . 1" t K . . .” I ".., . ’ ... E101" D Pathway I ,_ -'— 71'3/ \ D132 g.-. L ._ K Pathway A .1, \ ' "\ X /T"" \ ~ } Figure 5. 1: Proton uptake pathways in Rch0 The D and K-pathway are shown in blue arrows. The key residues are rendered as stick model. The waters in the pathway are rendered as yellow spheres. 117 and proton pumping efficiency (Pfitzner et al. 2000; Pawate et al. 2002; Han et al. 2006; Zhu et al. 2010), in some cases inhibiting and in others uncoupling activity. A chain of waters that hydrogen-bond to these hydrophilic residues was observed in high-resolution crystal structures of CcOs (Svensson-Ek et al. 2002; Qin et al. 2006; Koepke et al. 2009). The waters are in conserved positions in the crystal structures and serve an important fimction in proton uptake and proton pumping. The K-pathway was named after a conserved lysine residue in subunit I. Mutational evidence indicates that the K-pathway starts from glutamate E101 in subunit II, involves a water bound between S299 and K362, and another water at T359. The K-pathway ends below heme a3, where the Y288-OH and heme a3 famesyl-OH form a tight hydrogen bond that has been suggested to control the entrance of protons into the active Site (Xu et al. 2007; Sharpe and Ferguson-Miller 2008; Qin et al. 2009). Mutations in this pathway cause major loss of enzyme activity (Hosler, Shapleigh ,et al. 1996), but the remaining activity still appears coupled to proton pumping (Hosler et al. 1996; Adelroth et al. 1998; Zaslavsky and Gennis 1998), implying that this pathway is exclusively involved in substrate proton uptake. High-resolution crystal structures of two key mutants, D132A and K362M, would help us to further understand the function and mechanism of proton uptake pathways in Rch0. Thus these two mutant proteins were crystallized and are discussed in this Chapter. Both oxidized and reduced forms Of crystals of D132A and K362M were analyzed and studied. On-line spectroscopy on the single crystals of D132A and K362M was also performed to study the effects of X-ray radiation on the crystals during data collection. 118 2. Methods and results 2.1 Plasmid construction Two expression plasmids were constructed to express D132A and K362M proteins for crystallization. The pJL34 (for the expression of D132A) and pJ L35 (for the expression of K362M) are derived from plasmids pCH6l and pCH62 (from Dr. Carrie Hiser in our lab) by inserting a fragment containing a Short form of subunit IV gene (18 residues deleted from the C-terminus). The purpose of adding the short form of subunit IV was to improve the crystallization quality for the D132A and K362M, although later results showed that the addition did not have an effect on the crystallization of 4-subunit or 2-subtmit crystals. After construction, the plasmids were then conjugated into R. sph strain A1 A4 by using the method described in Chapter 2. 2.2 Protein expression and purification After conjugation, the R.sph strains containing D132A or K362M were grown and harvested as described in Chapter 2. The Spectra of membrane fractions of D132A and K362M are shown in Figure 5.2. D132A and K362M proteins were purified using the one-step Ni-NTA F PLC method described in Chapter 2. The membrane spectra showed that the constructs were successfully expressed. However, the expression levels of the CcOs from D132A and K362M are quite low compared with WT CcO and other mutants, making them difficult to purify and crystallize. 2.3 Crystallization of D132A and K362M Both 4-subunit and 2-subunit crystallization conditions were tried on the purified D132A and K362M proteins. Both forms of crystals could be found under appropriate conditions (Table 5.1). The crystals were analyzed and diffracted at the ANL synchrotron. Datasets 119 A: D132A 0.08 1 0.06 - 0.04 ~ 0.02 - 0 1 v -0.02 1 50.04 . . . . 500 550 600 650 700 Wavelength (nm) Absorbance 0.03 : . . 500 550 600 650 700 Wavelength (nm) A Figure 5. 2: Spectra of membrane fraction of D132A and K362M. The 605 nm peak in both D132A and K362M reduced minus oxidized spectra is very low compared to 550 nm peak. This suggests that although D132A and K362M protein were expressed successfully, the expression level of them is very low. 120 D132A K362M 4-subunit 6 pL of enzyme solution 6 pL of enzyme solution Crystals containing 10 mM Tris, pH 8.0, containing 10 mM Tris, pH 8.0, 50 mM NaCl, 0.20% decyl 50 mM NaCl, 0.20% decyl maltoside and 120 pM CcO was maltoside and 120 pM CcO was mixed with 6 pL of reservoir mixed with 6 pL of reservoir solution containing 100 mM solution containing 100 mM MES, pH 5.8, 23% PEG-400 MES, pH 5.8, 24.5% PEG-400 Best Resolution: 3.5A Best resolution: 3.7A 2-subunit 6 pl of enzyme solution 6 pl of enzyme solution Crystals containing 10 mM Tris, pH 8.0, containing 10 mM Tris, pH 8.0, 50 mM NaCl, 0.20% decyl maltoside and 120 pM CcO was mixed with 3 pL of reservoir solution containing 100 mM MES, pH 6.3, 29% PEG-400 and 3 pl Of crystallization additive solution containing 5% heptanetriol, 32 mM MgCl2, 1.3 mM CdCl2 and 0.026% dodecyl maltoside. 50 mM NaCl, 0.20% decyl maltoside and 120 pM CcO was mixed with 3 pL of reservoir solution containing 100 mM MES, pH 6.3, 27.5% PEG-400 and 3 pl of crystallization additive solution containing 5% heptanetriol, 32 mM MgCl2, 1.3 mM CdCl2 and 0.026% dodecyl maltoside. Best resolution: 2.15-2.5A Best resolution: 2.30-2.6A Table 5. 1: Crystallization conditions of D132A and K362M 121 D132A K362M 4-subunit 6 pL of enzyme solution 6 pL of enzyme solution Crystals containing 10 mM Tris, pH 8.0, containing 10 mM Tris, pH 8.0, 50 mM NaCl, 0.20% decyl 50 mM NaCl, 0.20% decyl maltoside and 120 pM CcO was maltoside and 120 pM CcO was mixed with 6 pL of reservoir mixed with 6 pL of reservoir solution containing 100 mM solution containing 100 mM MES, pH 5.8, 23% PEG-400 MES, pH 5.8, 24.5% PEG-400 Best Resolution: 3.5A Best resolution: 3.7A 2-subunit 6 pl of enzyme solution 6 pl of enzyme solution Crystals containing 10 mM Tris, pH 8.0, containing 10 mM Tris, pH 8.0, 50 mM NaCl, 0.20% decyl maltoside and 120 pM CcO was mixed with 3 pL of reservoir solution containing 100 mM MES, pH 6.3, 29% PEG-400 and 3 pl of crystallization additive solution containing 5% heptanetriol, 32 mM MgCl2, 1.3 mM CdCl2 and 0.026% dodecyl maltoside. 50 mM NaCl, 0.20% decyl maltoside and 120 pM CcO was mixed with 3 pL of reservoir solution containing 100 mM MES, pH 6.3, 27.5% PEG-400 and 3 pl of crystallization additive solution containing 5% heptanetriol, 32 mM MgCl2, 1.3 mM CdCl2 and 0.026% dodecyl maltoside. Best resolution: 2.15-2.5A Best resolution: 2.30-2.6A Table 5. 1: Crystallization conditions of D132A and K362M 121 280— 6::