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A .. v 00‘ . llolr I o. I» r. A ‘ ruU' ‘luAd,..[u up“. HI I! if ct... u‘ |v I t i... l. y wlIuOiiH ‘Il‘rlltt thin! I}. ‘I l’ i IIIIIIIIIIIIIIIII I\I\\\\\\\\\\\\\\\\\\\\\\\\\\ 3 1293 0091 This is to certify that the dissertation entitled KINETIC STUDIES OF CHARGE RECOMBINATION REACTIONS IN PHOTOSYSTEM II BY TRANSIENT ABSORPTION SPECTROSCOPY presented by Xingmin Liu has been accepted towards fulfillment of the requirements for Ph.D. Chemistry degree in W] W Major professor Date %7/fo. MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771 LIBRARY Michigan State University L PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE ll l. Jl —_7 L ll MSU Is An Affirmative Action/Equal Opportunity Institution (swims-9.1 KINETIC STUDIES OF CHARGE RECOMEINATION REACTIONS IN PHOTOSYSTEM II BY TRANSIENT ABSORPTION SPECTROSCOPY BY Xingmin Liu A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1990 9’5 7, w. ,L-//‘ /', 1's 3" ABSTRACT KINETIC STUDIES OF CHARGE RECOMBINATION REACTIONS IN PHOTOSYSTEM II BY TRANSIENT ABSORPTION SPECTROSCOPY BY Xingmin Liu The rates of charge recombination reactions of QA' and P‘680 in tris, NaCl, and NHZOH treated PSII membrane fragments have been measured under repetitive flash excitation by monitoring the time course of optical absorption changes that occur in the ultraviolet and near infrared spectral regions. In tris-inhibited membranes, the recombination reaction was stimulated by increasing the flash repetition rate; following NHZOH addition to these membranes and a preillumination regime, QA'lP’68O recombination was the major decay pathway for all flash repetition rate employed (0.2-10 Hz). In both preparations, however, the halftime of the charge recombination reaction between QA' and P’68O is approximately 230 us. In NaCl washed PSII, on the other hand, the recombination is faster, with a halftime of ~ 130 us. This difference confirms earlier EPR results and indicates that the presence of the manganese ensemble involved in water oxidation and the 33 KDa peripheral polypeptide influence the rate of the QA'/P*680 recombination reaction. Secondary electron transfer from Q,” to QB does not compete effectively with QA'/P*680 recombination in the inhibited, PSII membrane fragments used in this study. This behavior may result from release of plastoquinone during preparation of the membranes or from a Coulombic effect in which the positive charge on P’680 retards the QA' to Q8 electron transfer. The P‘680/P680 absorption difference spectrum measured in tris-washed PSII and NHZOH treated tris-PSII have nearly identical features. This suggests that NHZOH has no direct interactions with the reaction center P680; the inhibition site of NHZOH is probably located at Y2, at an auxiliary chlorophyll or, in the PSII reaction center polypeptides. To my parents, my wife, my teachers, and all friends of mine who have given me love, encouragement, and placed great hopes on me. iv ACKNOWLEDGMENTS I would like to thank Dr. Gerald T. Babcock for the independence and guidance he offered through this project. I would also like to thank Marty Rabb for his great assistance on the electronics, Dr. Tom Atkinson for his help on the critical points of programming, Dr. Dwight Lillie and Dr. Curtis Hoganson for their patience and time spent teaching and helping me with the computer interfacing. Finally, I would like to thank all my fellow colleagues in the laboratory for their friendship and assistance. TABLE OF CONTENTS Page LIST OF TABLES ..... ..................................... ix LIST OF FIGURES ......................................... x LIST OF SYMBOLS ......................................... xv CHAPTER I - INTRODUCTION Photosynthesis in Higher Plants .................... 3 Electron Transfer in Photosystem II ........... 7 The Structure of Photosystem II ..... .......... 12 Transient Absorption Spectroscopy in the Studies of Photosystem II .......... ......... 17 Reaction Center P680 ................ . ..... .... 18 Acceptor Side . 0 . 0 . 0 0 . . . 0 0 . 0 . 0 . . 0 . 0 0 0 0 0 0 0 0 0 . 0 0 0 19 Donor Side 0 0 0 0 0 0 0 0 0 . . 0 . 0 0 0 . . 0 0 . . . . . 0 0 0 0 0 0 0 0 0 0 0 2 2 Description of Work to be Presented ................ 24 References ...... ................................... 28 CHAPTER II - TRANSIENT ABSORPTION SPECTROSCOPY systemDeSj-gn 0.0.0.0000... ......... .00....000000000 36 vi Optical subsystem 00.000000000000000...00000000 38 Detecting and Signal Processing ............... 42 Computer Interface ................................. 48 DataAcquiSition 0.00.00.00.00.0000.00.00.00... 49 Timing contrOI 0000000000000...000.000.0000.... 55 Software Design .. ...... .... ............. . ...... .... 57 ProgramTASCI 0.0000.000.000.000.0000000000.... 58 Program DTDSP ................... ...... . ....... 60 References 000.0.0000000000000000...0 00000000 0 000000 63 CHAPTER III - THE CHARGE RECOMBINATION REACTIONS OF THE CHARGE SEPARATION STATE Q{/P"680 IntrOduction ......0......0...00.000.000.00000000... 64 Materials and Methods .............................. 69 Photosystem II Membrane Preparation ........... 69 Tris Treatment ................................ 71 NaCl Treatment ................................ 72 Hydroxylamine Treatment ....................... 73 Measurement Conditions . ....................... 73 Results ......... ........................ . ..... ..... 75 Excitation Saturation Profile ................. 75 Q{/P”68O Recombination in Tris-PSII .......... 77 QA‘/P*680 Recombination in NaCl Washed p311 . . . 83 QA’/P*680 Recombination iJIDHDOH Treated Tris-PSII .................... 85 C-SSO Bandshift ............................... 88 Discussion ... ........... ...... ............... . ..... 91 conCluSion 000.000.00.00. 0000000000000 . 00000 . 00000 00 96 vii References .0000.00.00....000000000000000.000000000. 98 CHAPTER IV - CHARACTERIZATION OF P+680/P680 AND Yz+/Yz ABSORPTION DIFFERENCE SPECTRUM IntrOduction 00.000000000000000000000 00000000 0.0.00 103 Materials and Methods ............................. 107 Results ........................................... 109 IV680/P680 Difference Spectrum ............... 109 Yf/Yé Difference Spectrum ...... ..... ......... 116 DiscuSSion 000000000000...0.00.00.00.000000.00.000. 121 conCIqu-on 0.0000000000000000000000000.00.000.00... 128 References 00000000000000.000000000000.00.0000.000. 130 CHAPTER V - SUMMARY AND FUTURE WORK Summary ........... ...... .......... ....... .......... 134 Future Work .. .......... . ........... . ..... ......... 137 References ..... ........... ..... ..... ....... ....... 140 APPENDIX A - SOURCE CODES OF PROGRAM TASCI ............. 142 APPENDIX B - SOURCE CODES OF PROGRAM DTDSP ............. 157 viii LIST OF TABLES CHAPTER II Page Table 1 Command Register . . . . . . . . . . . . ........... 52 Table 2 Status Register 53 CHAPTER III Table Halftimes of the Microsecond Decays . . . . 90 ix Figure Figure Figure Figure Figure Figure Figure 1-1 1-2 1-3 LIST OF FIGURES Page The electron transfer and proton transfer patterns of photosynthesis in higher plants ............. ............ 5 Z-scheme of electron transport in higher plants. P700 and P680 are the photochemical reaction centers of the photosystem I and photosystem II respectively. Pheo is a pheophytin molecule that acts as the primary electron acceptor of the P680. A is the primary electron acceptor of P700 and is presumed to be a Fe-S center ... .......... 6 The kinetics of electron transfer in photosystem II ............. ........... 11 A possible structure of photosystem II reaction center 00000000000000.0000000.... 14 Instrument setup ....... .................. 37 Optical layout of the transient absorption spectrometer used in the studies of the electron transfer in PSII (only relative positions are shown) ...... 40 The block diagram of the detecting and signal processing subsystem .......... 43 Figure 2-4 Figure 2-5 Figure 2-6 Figure 3-1 Figure 3-2 Figure 3-3 Pre-amplifier circuit. Iin comes from the PMT anode, Vout goes to the fourth order low pass active filter. The offset is controlled from the instrument panel. The operational amplifiers used are Burr Brown 3551 with 50 MHz GBP . ..... 45 The sample/hold amplifier. The two outputs, one is normal output (the upper output) for DC level measurement, another is DC subtracted output (the lower output) for the signal digitizer. Two variable resistors are used to balance the inverted signal .............. 47 The address map of the WAAG-1000 data acquisition board ..... .............. 50 Amplitude of the absorption changes at 325 nm as a function of the excitation flash energy in tris-washed PSII. [Chl]=196 pg/ml, optical path=0.14 cm, 2.5 mM K3Fe(CN)6 and K,Fe(CN)6 were added as the electron acceptor system .... 76 Microsecond time courses of the absorption changes at 325 nm (trace A) and 820 nm (trace B) with tris-washed PSII. [Chl]=215.7 ug/ml, optical path=0.14 cm, excitation flash repetition rate 5.8 Hz. 2.5 mM Fe(CN)6'3/Fe(CN)6‘2 were added as the electron acceptor system. The transient in the first few us was unreliable because of flash artifacts .... 78 Millisecond time courses of the absorption changes at 325 nm with tris- washed PSII (trace A) and NHZOH treated tris-PSII (trace B). [Chl]=215.3 ug/ml, optical path=0.14 cm, the time between the excitation flashes was 1.98 second. 2.5 mM I“e(CN)6'-”/12‘e(CNN;2 were added as xi Figure 3-4 Figure 3-5 Figure 3-6 Figure 3-7 Figure 3-8 Figure 3-9 the electron acceptor system. The transient in the first few us was unreliable because of flash artifacts .... 79 The dependence of the absorption changes at 820 nm on the flash repetition rate. Trace A is tris-washed PSII, trace B is NHZOH treated tris-PSII 81 The dependence of the absorption changes at 325 nm on the flash repetition rate in tris-washed PSII ...... 82 Microsecond time courses of the absorption changes at 325 nm (trace A) and 820 nm (trace B) in NaCl washed PSII. [Chl]=214.1 ug/ml, optical path=0.14 cm, excitation flash repetition rate 5.8 Hz. 2.5 mM Fe(cu);3/Fe(cu);¢ were added as the electron acceptor system. The transient in the first few us was unreliable because of flash artifacts .... 84 Microsecond time courses of the absorption changes at 325 nm (trace A) and 820 nm (trace B) with NHZOH treated tris-PSII. Experiment conditions were same as those in Figure 3-2. The transient in the first few us was unreliable because of flash artifacts .... 86 The dependence of the absorption changes at 325 nm on the repetition rate of the excitation flash in NHgfli treated tris-PSII ........................ 87 Microsecond time courses of the C550 band shift in tris-washed PSII (trace A) and NHZOH treated tris-PSII (trace B). [Chl]=315.7 ug/ml, 2.5 mM Fe(CN);3/ Fe(CN)6'2 were added as the electron acceptor system ................... ....... 89 xii Figure 3-10 Figure 4-1 Figure 4-2 Figure 4-3 Figure 4-4 Figure 4-5 The scheme of the electron transfer pathways in tris-washed PSII ... .......... 93 Absorption difference spectrum of the initial amplitudes of the total absorption changes after 5 us following the flash excitation in tris-washed PSII. [Chl]=215 ug/ml, optical path=0.14 cm, excitation flash repetition rate 5.8 Hz. 2.5 mM Fe(CN);3/Fe(CN)§‘ were added as the electron acceptor system ........... ..... 111 Absorption difference spectrum of the 230 ps decay phase in tris-washed PSII ........................ 112 Absorption difference spectrum of the reaction center P’680/P680 in tris-washed PSII (Open circle, continuous line) and the spectrum of Chla"/Chla in CI-IZCI2 (dash line) . . . . . . . 113 Absorption difference spectrum of the reaction center P’680/P680 in tris-washed PSII (open circle) and iJlDHEOH treated tris-PSII (open triangle). The NHZOI-I treatment was performed with the addition of 2.0 mM IHEOH and 60 pre-flashes before the measurements were taken in tris-washed PSII. The experiment conditions were the same as in Figure 4-1 ............... 115 Absorption difference spectrum of the millisecond decay phase in tris- washed PSII (open circle) and in DHMOH treated tris-PSII (open triangle). [Chl]=215 ug/ml, the time between excitation flashes was 1.98 second. 2.5 mM Fe(CN)6'3/Fe(CN)6"’ were added as the electron acceptor system ......... 118 xiii Figure 4-6 Figure 4-7 Figure 4-8 Figure 4-9 Absorption difference spectrum of the ijYi in tris-washed PSII. The spectrum is obtained by subtracting the QA'/QA contributions from the spectrum in Figure 4-5 (open circle). The overlayed spectrum (open triangle) is the absorption difference spectrum of the phenoxy radical Ter' in water ...... 119 Absorption difference spectrum of the millisecond decay phase in NHgni treated tris-PSII after subtracting QA'/QA spectrum from the spectrum in figure 4-5 (open triangle) . ..... ..... 120 The electron transfer and hydrogen shift of the electron donor Y2 during the electron transfer reactions in PSII. Here B represents an amino acid residue that forms a hydrogen bond with the tyrosine .................. 126 The possible chemical reactions of hflQOH with the phenol group of the tyrosine residue of Y2 in PSII . ......... 127 xiv ADP ATP BChl Chl CP Cyt DCBQ DCMU DTDSP EDTA EPR Hepes Mes NADP LIST OF ABBREVIATIONS AND SYMBOLS adenosine diphosphate adenosine triphosphate bacteriochlorophyll chlorophyll chlorophyll protein cytochrome 2,6—dichloro-p-benzoquinone 3(3',4'-dichlorophenyl)-1,1-dimethylurea data treatment and display program ethylene diamine tetraacetate electron nuclear double resonance electron paramagnetic resonance 2-hydroxyethyl-1-peperazineethanesulfonicacid 4-morpholine-ethanosulphonic acid nicotidamide adenine dinucleotide phosphate nuclear magnetic resonance XV OEC P680 PC Pheo PQ PSI PSII s (n=0-4) I'I TASCI Tris oxygen evolving complex reaction center chlorophyll in photosystem II phycocyanin pheophytin plastoquinone photosystem I photosystem II quinone acceptor in photosystem II quinone acceptor in photosystem II states of charge accumulating system associated with the oxygen evolving complex transient absorption spectrometer computer interface program tris(hydroxymethyl) amino methane tyrosine residue at 160 position of D1 polypeptide of photosystem II reaction center complex, functions as an electron donor to the reaction center P‘68 0 tyrosine residue at 161 position of D2 polypeptide of photosystem II reaction center complex, it does not participate in electron transfer reactions under physiological conditions xvi CHAPTER I INTRODUCTION Photosynthesis is the process whereby solar radiation is captured and converted into chemical energy. It takes place in plants, algae, and some bacteria. It provides most of the energy we use today. All our foods are direct or indirect products of photosynthesis, and the presence of oxygen in the earth's atmosphere is a direct result of the photosynthetic reactions of higher plants and algae. Photosynthesis starts with the absorption of a photon by one of the antenna pigment molecules that include species such as chlorophylls, bacteriochlorophylls, carotenoids and phycobilins, which are organized in large, light-harvesting or antenna complexes. The excitation energy is then transferred from one pigment molecule to another by means of long range dipole-dipole interaction [1,2], until it reaches a specialized (bactero)chlorophyll complex, presumably a dimer [3], called the reaction center. There the photochemical reaction takes place, in which the excited (bacterio)- chlorophyll undergoes charge separation in which an electron 2 is transferred from the excited (bacterio)chlorophyll to an acceptor molecule. The electron is then quickly transferred to a secondary acceptor and the oxidized chlorophyll is quickly reduced by a donor molecule so that the reducing entity and oxidizing entity are further separated in order to increase the quantum: efficiency’ of the forward electron transfer and reduce the chance of back reaction- The separated charges are subsequently further stabilized by a series of dark electron transport reactions. Eventually the positive charge at the donor side of the photochemical reaction center is neutralized by an electron from an oxidizable substrate HZA where, in higher plants and algae, A is oxygen and.in.photosynthetic bacteria A.is a highly reduced organic molecule [4]. The negative charge at the reducing side of the photochemical reaction center is ultimately used in the carbon dioxide fixation process in plants, algae and most photosynthetic bacteria, although some photosynthetic bacteria may not fix carbon dioxide [5,6]. The consequences of this process are that solar energy is finally stored in organic compounds as chemical energy and substrate HZA is oxidized as a by-product. The electron transfer process in photosynthesis is almost 100% efficient in the sense that for each photon absorbed one electron is transferred from substrate to its acceptor without any significant back reaction during its transport. This 3 remarkable efficiency of electron transfer is achieved with the highly organized membrane protein complexes of the photosynthetic membrane so that the electronic coupling and reorganization energy of forward electron transfer appear to be optimized» The study of this electron transfer process not only' gives us better understanding of the mechanism of photosynthesis but also provides us with very useful clues as to how to store the solar energy more efficiently. Photosynthesis in Higher Plants Photosynthesis in higher plants takes place in a special cell organelle, the chloroplast. This contains an intricate lamellar system, composed of flattened membrane sacs (thylakoids), which enclose the so-called lumen. In higher plants, the thylakoids are usually arranged.in Stacked (grana) and unstacked (stroma) regions [7]. The primary reactions of photosynthesis take place in these membranes. The overall reaction in photosynthesis in plants can be written as: hv H20 + co2 ———> 1/6 (C6H1206) + 02 AG = 484 Kcal/mol Photosynthesis in plants and algae differs from photosynthesis in photosynthetic bacteria in two aspects (1) Because of the high free energy requirement for photosynthesis 4 in plants, AG = 484 Kbal/mol, a single quantum of visible light has not enough energy to drive the reaction. Thus, in plants there are two photochemical reaction centers working in series so that each. electron 'transfer' is driven by two photons. In photosynthetic bacteria, which do. not oxidize water, there is only one photochemical reaction center. (2) In plants the main pigments are chlorophylls. In photo- synthetic bacteria the main pigments are bacteriochlorophylls. In plants the two photochemical reactions are performed by photosystem I (PSI) and photosystem II (PSII). The general patterns of electron and proton transfer in these photosystems in higher plants are shown in Figure 1-1. Each photosystem consists of a reaction center, an associated antenna system with one to four hundred pigment molecules (chlorophylls and carotenoids), and several redox active components that serve as electron donors and acceptors. IPSII is responsible for the oxidation of water and transfers electrons to PSI through the plastoquinone molecules in the PQ pool, while PSI transfers the electron to its final acceptor NADP“, which is used to reduce C02. Coupled with the electron transfer from PSII to PSI, protons are also transferred across the thylakoid membrane from the stroma side to lumen side. The proton gradient generated by this proton transfer is used to synthesize ATP from ADP by the membrane bound ATP synthase. In general, the electron transfer process of photosynthesis in outside (stroma) 4H+ 2NADP+ 2NADPH am my 4% ATP ‘m ATPase 0 + 2H20 02+ 4H+ 8“ . inside (lumen) Figure 1-1 The electron transfer and proton transfer patterns of photosynthesis in higher plants NADP / PSI A< P700 Q ///////}7 H20 PSII \ Pheo=< P680 -1.0 -O.5 0.0 +0.5 {+1.0 Em in Volts Figure 122 Z-scheme of electron transport in higher plants. P700 and P680 are the photochemical reaction centers of the photosystem I and photosystem II respectively. Pheo is a pheophytin molecule that acts as the primary electron acceptor of the P680. A is the primary electron acceptor of P700 and is presumed to be a Fe-S center. 7 higher plants involves a series electron donors and acceptors of varying redox potential. If the electron transfer diagram is drawn with redox midpoint potential as the Xkaxis, the diagram looks like a letter Z, thus it is called the Z-scheme for photosynthetic electron transfer (Figure 1-2). Electron Transfer in Photosystem II The electron carriers in PSII consist of a manganese cluster (four manganese ions), intermediate electron carrier Y2, reaction center P680, which has its absorbance bleaching maximum at 680 nm, primary acceptor Pheo, and plastoquinone acceptors QA and 93° Upon light excitation, the reaction center P680 (presumably a dimer of chlorophylls) is excited and donates an electron to its nearest acceptor Pheo (pheophytin a) in 3 ps [8]. The charge Separation is stabilized in 300 ps [9] by'a secondary electron transfer from Pheo’ to a strongly bound plastoquinone molecule called QA. The reduced quinone acceptor'Q{ can be reoxidized in a variety of ways. In intact PSII membrane particles in which the QB site is not empty or damaged, or in chloroplasts, Q“ is oxidized by a secondary plastoquinone molecule called QB in about 200 us [10,11]. The QA' to Ql3 electron transfer can be inhibited by a number of commercial herbicides, such as DCMU (3 (3 ' , 4 ' -dichlorophenyl) -1 , 1-dimethylurea) , which are commonly used in the studies of PSII. In some conditions, q; can be 8 oxidized in approximately 40 us by the oxidized non-heme iron located between QA and QB [12,13] . QA' can be also oxidized by exogenously added acceptors such as 2,5—dichlorobenzoquinone (DCBQ) or ferricyanide, although it is unclear in the ferricyanide case whether 0; is oxidized directly or indirectly [14]. An additional means of 0" oxidation is importance to the research in this thesis, namely, backreaction or charge recombination with P”680 or Yz“. This reaction takes place when the normal electron transfer on the donor side of PSII is retarded or inhibited. The QA’ oxidation by charge recombination is the main topic of this dissertation, and it will be discussed in the following chapters in greater detail. The water splitting reaction is the least understood part of photosynthesis in higher plants. Although significant progress has been made during the last decade, water oxidation is still an unsolved problem. In order to oxidize water, four oxidizing equivalents are required: 4hv + - ZHZO —-——> 02 + 4H + 4e The photochemical reaction that takes place in PSII is a one electron reaction. How this one electron reaction is coupled to the four-electron oxidation of water is a key point in understanding the water splitting process. The single 9 turnover flash experiments done by Joliot, Kok, and their coworkers [34-36] showed that each oxidizing equivalent is stored in a complex until four have been accumulated. Only when this is accomplished is oxygen released. This is the so-called Kok model 02 < 21120 In this scheme, S represents the state of the oxygen evolving complex, and the subscripts represent the oxidizing equivalents stored in the complex. Although this model has nothing to say about the nature of the S-state complex, it predicts the existence of relatively stable higher S-state intermediates. Manganese has long been known to be essential for water splitting activity [37], as a substantial body of work [37-40] indicates that four manganese are associated with the S-state complex. Transient absorption experiments in UV region [14] , the multiline EPR signal [41-44], and K edge manganese X-ray absorption [45] strongly indicate that the manganese cluster is directly involved in the electron transfer reaction between water and Y;'and that it undergoes valence changes during the S-state transitions. The redox potentials involved on the donor side of PSII 10 are extremely high and easily lead to non-physiological oxidation products such as chlorophylls [15,16], cytochrome b5” [17,18], and carotenoid [19]. Thekinetics of electron transfer on the donor side of the PSII are different for the four different redox states of the oxygen evolving complex. Under normal physiological conditions, the oxidized PSII reaction center P‘680 is reduced by a component called Y2, which has been identified as a tyrosine residue of the D1- polypeptide at the 160 position [20,21]. The electron transfer rate from Y2 to P‘680 is highly dependent on the oxidation state of the oxygen evolving complex, pH in the buffer, and polypeptide composition. At physiological pH values, the electron transfer takes place in 20 ns in lower oxidation states of the oxygen evolving complex (So and S1) and is biphasic in higher oxidation states of the oxygen evolving complex (S2 and S3) with halftimes of 50 ns and 250 ns [22,23] . At low pH, the electron transfer is monophasic for all oxidation states of the CBC: the halftime is 40 ns in So and S1, and 250 ns in $2 and S3 [24]. For tris-washed PSII, in which the manganese cluster and 3 peripheral polypeptides are released, the rate of the electron transfer is much slower, being a few microseconds [25,26]. Photosystem II contains a second tyrosine residue, called YD, located on D2-polypeptide at the 161 position [20,27] . It has the same EPR spectrum as Y2, but the decay kinetics and 11 "a QB N External Acceptor: me a 0') 4H‘+ 0, If: 9:: Y2 20‘900 L. ZHZO m \ P +68 0 Figure 1-3 The kinetics of electron transfer in photosystem II interactions with the manganese cluster are different [28-30] . Normally, this tyrosine residue is oxidized and does not participate in electron transport. It is, however, involved in maintaining the system in the S1 state in the dark [31,32] and preventing over-reduction of the oxygen evolution complex [33]. A potential function in photoactivation of the manganese cluster has also been proposed [27]. It is generally agreed that the manganese cluster functions between substrate water and the intermediate 12 electron carrier, Y2, and that it functions as a four-electron gate. It stores positive charge equivalents prior to the four electron oxidation of two molecules of water. In addition, it is likely that manganese acts as the binding site for the water’molecules that are oxidized. For intact PSII membranes, where the manganese cluster is fully functional, Y; is reduced by the manganese cluster. The rate of this electron transfer is highly depend on the oxidation state of the manganese cluster. In the 80 state, Yz" is reduced in 30-70 us, in the S. state, it is reduced in 50-110 us, in the 32 state, it is reduced in 60-350 us, and in the S3 state, the reaction is further slowed down to one millisecond [46-50]. When the manganese cluster is destroyed, such as by tris washing, which releases manganese and peripheral polypeptides from the OEC [51-52], Y; can be reduced by an eXternal donor or by charge recombination with Q{. These reactions will be discussed in detail in chapter III and chapter IV. The structure of Photosystem II Photosystem II of higher plants is made up of a cluster of at least nine polypeptides (Figure 1-3), six of which are intrinsic, membrane-spanning polypeptides, and three of which are extrinsic, membrane associated polypeptides. The exact locations of the various electron transport components discussed above are not known yet. No single crystal 13 structure of PSII has been obtained, thus there is no detailed structural information available as there is in the photosynthetic bacteria case [53]. Figure 1-3 shows a possible model of PSII structure, which comes from the assumption that the structure of PSII is similar to the structure of photosynthetic bacteria, though the justification of the assumption is still an open question. Recent results from Yocum's laboratory have strongly challenged this assumption [54]. The six intrinsic polypeptides of PSII include the following: two large chlorophyll-binding polypeptides with apparent molecular masses of 47 and 43 kDA, called CP47 and CP43 respectively, that serve as core antenna subunits; two distinct polypeptides with molecular masses of approximately 32 kDA, named D1 and D2, that bind to each other and are the essential parts of the PSII reaction center; and two other small polypeptides (5 and 10 kDA) that are involved in the binding of the cytochrome b559 heme group. The locations of the reaction center and other electron transfer carriers of PSII within these polypeptides are not clear yet. In early work, the CP47 polypeptide was proposed by several groups to be the site of P680 and pheophytin a binding based upon fluorescence [55], pigment composition and spectral change [56-58]. However, because of the homology in the amino acid sequences of the DI and D2 polypeptides with 14 43 32 32 1o 6 { 47 QA Fe“ Q! ‘ Cytb“, Phe o Pheo Hmo \ Y2 Y0 23 33 Figure 1-4 A possible structure of photosystem II reaction center 15 the L and.M subunits of the reaction center of purple bacteria [53], it was suggested that D1 and D2, rather than CP47, might provide the binding site for the P680 and pheophytin a [59-61]. Recently, evidence for the binding of pigments to D1 and D2 polypeptides has been provided by Nanba and Satoh [62]. They successfully isolated a complex consisting of the D1 and D2 polypeptides and cytochrome b559 from spinach PSII particles with 4-6 Chl a, 2 Pheo a, and 1 B-carotene molecules. This complex shows photochemical activity, as indicated by light induced absorbance changes that could be attributed to the photo-reduced Pheo a, and the formation of a spin-polarized triplet at low temperature that is characteristic of all reaction centers [63]. But the absence of plastoquinone in Nanba and Satoh's preparation indicates that the electron acceptor QA and Q8 are lost during the isolatiOn procedure. This raises the question as to whether the pigment composition obtained in the Nanba's particle really reflects the true pigment composition in PSII reaction center. Recent results from chum's laboratory indicate that more pigments, 10-12 Chla, 2-3 Pheo a, along with 2 Cyt b559, are bound to the reaction center complex of PSII [54]. There is also some evidence indicating that CP47 and CP43 not only serve as sub- antennae but also play a role in binding quinone molecules and the extrinsic 33 kDA polypeptide [63]. The three extrinsic polypeptides with molecular masses of 16 17, 23 and 33 kDA are associated with the inner surface of the thylakoid membranes and play important roles in oxygen evolution in higher plants [64,65]. At first it was assumed that these polypeptides were directly involved in the oxidation of water, but more recent evidence has shown that their main function is to shield and stabilize the manganese cluster. Each of the three polypeptides has now been shown to be functionally replaceable with high concentrations of chloride and/or calcium ions. The 17 kDA polypeptide is replaced by 5 mM Cl'. The 23 kDA polypeptide is replaced by 5 mM Ca" and 30 mM Cl'. In the absence of the 33 kDA polypeptide oxygen evolution can occur, although at a slower rate, when 200 mM Cl' is present [66]. Ca“ must also be present to overcome the absence of the 23 kDA in these preparations. Thus, it is thought that the extrinsic polypeptides play a role in improving the reaction center capacity for binding Ca+ and Cl' ions. In addition, they play a structural role in protecting the manganese cluster from attack by external reducing agents. It is possible that the 33 kDA may provide some ligands directly to the manganese cluster that are replaced by C1' when the polypeptide has been removed [67]. The location of the manganese cluster remains one of the most speculative parts of the PSII structural model. .Although the three extrinsic polypeptides are unlikely to be the major 17 sites of manganese binding [68], none of the six intrinsic polypeptides can be strictly ruled out as the binding polypeptide [69]. Based on mutagenesis experiments on the D1 polypeptide with an unprocessed C-terminal extension [70], photodestruction of the reaction center [71], and cross- linking studies of the complex [72], the prevailing view is that the manganese cluster is more likely to be situated in the D1/D2 reaction center polypeptides. Transient Absorption Spectroscopy in the studies of Photosystem 1; Transient absorption spectroscopy has been the oldest but the most useful physical technique to study photosynthesis. Although it does not have the high spectral resolution and selection that ESR and ENDOR do, it has better time resolution capability. Rapid development of laser technology has brought transient absorption spectroscopy to preeminence in studying rapid kinetic processes. The picosecond and sub-picosecond laser spectroscopy developed in the last few years has pushed the time resolution of transient absorption spectroscopy close to the theoretical limit. This kind of time resolution is impossible for other kinds of spectroscopies such as ESR, NMR and ENDOR. Transient absorption spectroscopy is a non- destructive technique, and samples used in the measurements are usually closer to their physiological conditions than 18 those used in other spectroscopies, which reduces the chances of interferences from non-physiological sample conditions. Almost all redox-active components of photosystem II have absorption changes upon oxidation or reduction following the excitation flash. Thus, they are detectable by transient absorption spectroscopy. Most of the kinetic data on electron transfer in PSII has been obtained by this technique. The spectral overlap of different components, which causes difficulty in interpreting the results, can be greatly improved in transient absorption spectroscopy, because oxidation or reduction of different components may take place on different time scales. Reaction Center 9680 The reaction center of PSII was detected as a flash induced absorption change attributable to chlorophyll a oxidation [73]. Its bleaching maximum is close to 680 nm and it thus was designated P680. The fluorescence of the light harvesting chlorophylls interferes with measurements at this wavelength, however, and many kinetic studies of IS680 have been done by measuring the smaller broad absorption increase at around 820 nm [74,75]. In the ultraviolet and visible region, because of the interferences from other components and strong absorption of samples, investigation of the P680 spectrum has not been done 19 as intensively. The first detailed P‘680/P680 difference spectrum in the UV/Visible region was published recently by Gerken and coworkers [76]. The UV/Visible region is a very important region because structural or environmental changes may be detected fairly easily in this region. The appearance kinetics of P’680 following light absorption was impossible to monitor few years ago. Recently, however, Wasielewski's laboratory used 500 fs time resolution to resolve this rise time as 3 ps by transient absorption spectroscopy. The data show that P680 donates an electron to a pheophytin a molecule directly without detectable electron transfer participation of the accessory chlorophyll [8]. The P’680 decay can be detected optically at 820 nm. Under physiological conditions, P”680 is reduced on a time scale of 50 to 250 ns depending on the S-state. When the normal electron transfer is inhibited (e.g. by tris-washing, salt washing, NHZOH treatment, detergent treatment, or extremes pH), I”680 reduction is retarded into the microsecond range and can be easily detected optically. Acceptor Bide Photoaccumulation of reduced pheophytin in PSII detected by its characteristic absorption changes under reducing conditions was first reported by Klimov et al.[77]. It was proposed that Pheo might play a role as an electron carrier 20 between the reaction center P680 and the plastoquinone acceptor ri analogous to that of BPheo in purple bacteria. Supporting evidence came from.the observation of a split Pheo' EPR signal [78] and from the observation of a characteristic spin-polarized triplet signal from P680 [79]. The difference spectrum of Pheo‘lPheo is similar to that of P’680/P680 in the red and near infrared region, but the small bleaching at 540 nm and 505 nm are typical of Pheo reduction [80,81]. The most direct proof that Pheo acts as an electron carrier between P680 and QA comes from the direct observation by transient absorption spectroscopy of Pheo reduction with sub- picosecond time resolution. It has been shown that Pheo is reduced in 3 ps [8] by P680 and reoxidized in 300 ps as it 'transfers the electron to acceptor QA [9]. A great deal of work on Q, has been done by using absorption and fluorescence spectroscopy. It has been identified as a firmly bound plastoquinone molecule by absorption spectroscopy and extraction/reconstitution experiments [82]. (2M is reduced to a semiquinone anion by Pheo' in about 300 ps. The absorption difference spectrum reflecting this transfer is characterized by the appearance of the semiquinone anion absorbance at 325 nm and the disappearance of the oxidized quinone at 265 nm [14]. In the visible region of the spectrum, a second semiquinone absorption band is observed between 400 nm and 450 nm, but 21 here the spectrum is superimposed on changes due to electrochromic wavelength shifts of the absorbance of chlorophyll and pheophytin molecules. These shifts are caused by the charge on 0‘, and probably involve two pigments in the reaction center, chlorophyll a and pheophytin a. The pheophytin a bandshift around 545 nm is important for diagnostic reasons, since it occurs in a wavelength region in which. the absorbance changes can, be :measured relatively easily. Moreover, QA is the only quinone that induces a significant shift of this pigment upon reduction [11,83]. Reduced QA‘donates an electron to another plastoquinone acceptor called QB in about 200 us [10,11] in intact PSII membrane particles. The reduction of QI3 to 03' gives rise to absorption changes in the UV that are similar to those observed when QA is reduced. The spectrum shows characteristic features of plastosemiquinone formation [14]. The 08' minus QB optical spectrum is similar in many respects to that of Q,” minus QA but the bandshifts in the blue and green parts of the spectrum are different [84], indicating that Q{ is different from Q; in terms of its proximity to the electrochromically responding pheophytin. The reduced 0,, remains bound to the QB-site [85] until a second electron from Q", produced in a second photoact, reduces it to a doubly reduced quinone. The doubly reduced Ql3 (QB'Z) is protonated to form the hydroquinone QBH27 it then leaves the binding site on 22 the reaction center and becomes part of the membrane plastoquinone pool. This mechanism of plastoquinone reduction exhibits a strong period two oscillation in UV absorption upon flash excitation. Thus, extreme care must be taken when analysizing the flash number dependence of absorption in the UV region. Donor Bide Although the electron transfer rate from Y2 to reaction center P‘680 is strongly dependent on the S-state, ranging from 20 ns to 250 ns, its absorption difference spectrum appears to be very similar in all these S-states [11,14,23]. It consists of absorbance increases around 260 nm and 300 nm and of a chlorophyll a bandshift around 435 nm. Because of the strong overlap of the absorption difference spectrum between QA'/QA and Elf/3!z in the UV region, where the major peaks of YZVYZ are located, care must be taken when the data are analyzed. However the EPR signal of Z+ is relatively easy to detect, so most studies of 312+ to date have been done by EPR rather than optically. Transient absorption spectroscopy provides an excellent tool to study S-state transitions, since other techniques, such as EPR and EXAFS that require cryogenic temperature, can not be done at physiological temperature. The kinetics of S-state transitions can be studied with single turnover 23 flashes with dark adapted PSII particles, and the period four oscillation of absorbance changes, which corresponds to four different S-state transition, can be observed in the UV region [46,86]. In dark adapted PSII, about 75% of the reaction centers are in the S1 state. The first flash induces the S1 to S2 transition, which produces an absorbance increase at 362 nm with a half time about 40 us [46]. The second flash induces the S2 to S3 transition with an absorbance increase similar to the first flash with a half time of about 100 ps. The third flash, which induces the S‘,,-(S,.)-So transition and is coupled with oxygen evolution, reverses the absorbance increases of the first and the second flashes and resets the system to the Sn state with a half time about 1.5 ms. The fourth flash, which induces the S0 to S1 transition, shows a very small absorbance increase with a halftime about 50 us [46] . Experiments done at different wavelength show very similar results [87]. The absorption difference spectrum of S-state transitions has a peak around 300 nm with extinction difference coefficients on the order of 4500-6000 chmq. Above 400 nm, absorption changes directly related to the manganese cluster are very small. The absorption difference spectrum of 8113: S2 transition and S2 to S3 transition are very similar. Both are tentatively assigned to a Mn(III) to Mn(IV) transition based on the experiments of multiline EPR signal [41,88-90], 24 XANES K-edge [91], and NMR proton relaxation rate [92]. The small absorption changes of the S0 to S1 transition are assumed to be due to a Mn(II) to Mn(III) transition based on other techniques [93]. Qescription of Work to be Presented In intact PSII membranes, the forward electron transfer is carried out very efficiently and the quantum yield is very close to J” This high efficiency of electron transfer is achieved by rapid reduction of the P‘680 by its physiological donor Y2 in less than 250 ns [22]. Certain chemical treatments, which inhibit oxygen evolution, greatly increase the lifetime of P“680 so that the charge recombination reaction of the state Q{]P"680 is extended into the microsecond time scale. The time evolution of the state Q{/P"680 is highly depended on the micro-environments of the electron carriers and the presence of inhibitors. Gerken and coworkers [76] studied the charge recombination reactions of Q{/P”680 in tris-washed PSII in cyanobacterium. They found three decay phases with.halftimes of 170 us, 800 ps and 6 ms, and attributed the multiphasic kinetics to a distribution of different structural states of the reaction center proteins. Ford and Evans [94] observed that, after NHZOH inhibition, the sub-microsecond decay phase of P‘680 in PSII was transferred into the microsecond time range. The 25 decay was biphasic with halftimes of 90-150 us and 600-900 us. Both phases were attributed to charge recombination between QA' and P’680. They explained the different kinetic phases in terms of different charge states of other redox-active components in the reaction center complex. Other inhibitory treatments include trypsination [95], which retards P"680 reduction to 200 us and may be relieved by the addition of Ca2+ cations, and addition of acetate [96] , which slows the forward reduction of P’680 by almost four orders of magnitude and the back reaction with QA' five-fold, were explained as modifications of the micro-environment of PSII. The wide differences of decay pathways and decay rates of the charge separated state, QA'/P"680, under different treatments raises interesting questions as to how the time evolution of QA'/P’680 is modified by these treatments and as to what information we can obtain by monitoring the time courses of the state QA'/P*680 following these different treatments. In the experiments described in chapter III, we used the transient absorption spectroscopy technique, described in chapter II, to monitor the decay time course of QA'lP’680 with tris, NaCl, and NHZOH treated PSII. Our results show that the charge recombination reactions can be observed in these inhibited PSII preparations. The halftimes of the charge recombination reaction are similar in tris-washed PSII and NHZOH treated PSII, but it is faster in NaCl-washed PSII. 26 These differences are attributed to the different micro- environments at the donor side of PSII. The QA’ to Q3 electron transfer, which has not been analyzed fully in prior studies, may not occur in these samples because of the positive charge on P’680 or the modification of Q8 site. An obvious way to identify the components of the electron transfer reactions and the effects of different treatments by transient absorption spectroscopy is to obtain the absorption difference spectrum of the individual components. The first full absorption difference spectrum of QA’/QA was obtained by Van Gorkom [82] and identified as arising from a plastoquinone molecule because of its similarities to the in vitro spectrum of plastoquinone. Dekker and coworkers [14] obtained detailed absorption difference spectra of Q,"/QA and Eff/Yz in tris- washed.PSII by analysislof flash-induced absorption changes in the millisecond time range. The absorption difference spectrum of P’680/P680 in the UV range has not been studied as extensively as that of QA'/QA and Yf/Yz because of its very short lifetime. The first detailed P‘680/P680 absorption difference spectrum in the UV-Visible region was obtained very recently by Gerken et. a1. [76] in tris-washed PSII from cyanobacterium Synechococcus sp. by measuring the charge recombination reactions between QA' and P‘680. They found that the P’680/P680 absorption difference spectrum is dominated by a very sharp bleaching around 434 nm, which resembles the in 27 ‘vitro difference spectrum of Chlaf/Chla. The absorption difference spectrum can be used to identify the various components as well as their micro-environments. Weiss and Renger [95] claimed that the difference spectrum of I”680/P680 was affected by the trypsination at pH 7.5 or by incubation with NHgfli. In the work described in Chapter IV, we measured the absorption difference spectrum of F”680/P680 and Y;]Y§ by monitoring the decay kinetics of Qp/PV680 under different treatments in tris-washed PSII. The results we obtain support a model in which the microsecond decay phase comes from the charge recombination reaction between Q{ and 1’680 and the millisecond phase comes from electron transfer between Q{ and Y{. The difference spectra we obtained with IUMOH treated tris-PSII show that the spectrum of PV680/P680 is not affected by the NHZOH treatment, contrary to the claim by Weiss and Renger [95], but that the spectrum of Y{]Y, is dramatically modified by NHZOH treatment. We conclude that the NHZOH inhibition site is closer to Y2 than to the reaction center P680 site. 10. 11. References T. Forster (1948), Ann Physik., 2, 55 R. 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Acta 1985, 807, l Weiss, W. and Renger, G. Biochim. Biophys. Acta 1986, 850, 173 Bock, C.H., Gerken, S., Stehlik, D. and Witt, H.T. FEBS Letpp 1988, 227, 141 CHAPTER II TRANSIENT ABSORPTION SPECTROSCOPY It.has been.42 years since the first spectrometers working in the ultraviolet and visible regions of the spectrum came into'general use [1], and over this period they have become an extremely important analytical instrument in many chemical and biological laboratories. Absorption spectrometry is a non- destructive technique and most measurements can be carried out at physiological temperature, this makes it a suitable technique to study biological system. Transient absorption spectroscopy measures the time evolution of the absorbance changes of a system after excitation. It has been widely used to study kinetics of chemical reactions. IRapididevelopment of laser technology has brought the transient absorption spectroscopy to preeminence in studying ultrafast reactions with picosecond or even sub-picosecond time resolution, which no other spectroscopy can match. Transient absorption spectroscopy has been heavily used in the studies of the electron transfer reactions of photo- synthesis. In the experiment, the probe beam is usually kept 35 36 very weak so that it will not cause extensive photochemical reaction. The sample is excited by an intense flash of short duration and the absorbance changes after the flash are monitored by the probe beam. The time course of the absorbance changes and its dependence on wavelength provide important information on the electron transfer reactions taking place and the components involved in the reactions. System Design Because the transient absorption system is designed specifically for the study of electron transfer reactions in photosynthesis, special considerations must be taken. First, the probe beam power must be kept low enough so that it does not cause significant photochemical reaction; Second, the optical path must be designed in a such way that fluorescence from the sample is blocked from reaching the detector; Third, because the absorbance changes to be detected are small compared with the background, the DC component of the signal must be subtracted out before it can be input into the analog to digital converter. The original system was built by D. Lillie [2], who interfaced. a NICOLET 1074 (Fabri Tek, equipped. with an SD-72/2A 9-bit A/D converter and a SW 71A sweep controller; Madison, WI) to a DEC (Digital Equipment Corporation) POP-11 minicomputer. Because of frequent failure of the hardware and .msumm unmasuumcH HIN ousowh postage Q\< . UAHH 3653688 cos; j? Interface 37 souumsh sees; nus—m some; 1%H\ _ 1 new; QE< _ - Iona — Ema 2A / .‘I seaceounoocs ou< ‘IJ‘ .. . l _ 38 inconvenience of using the PDP-ll minicomputer to control the instrument, it was rebuilt by me with a new data acquisition board (WAAG-1000 from Marckrich Corp.) and timer board (CTM-05 from Metrabyte Corp.), and interfaced to an IBM/AT compatible Zenith 248 personal computer. This made it possible to integrate the new system with the laser facility located in the chemistry department at Michigan State University. The system (Figure 2-1) consists of three:major subsystem: (1) the optical subsystem, which includes the probe light source, monochromator, silica lens, and cutoff filters; (2) the detecting and signal processing subsystem, which includes the photomultiplier tube, signal pre-amplifier, sample/hold amplifier, and fourth order low pass filter; and (3) the computer interface, which includes analog to digital conversion board, five channel timer board, driver circuit and software interface. The excitation flash is provided by a NszAG pulsed laser (DCR-l or DCR-2 from Quanta-Ray) at 532 nm (frequency doubled) or at 355 nm (frequency tripled) with a pulse width of about 10 ns. The laser is triggered externally by the computer. Optical Subsystem The optical subsystem was built by D. Lillie originally, but modification has been made in the following aspects in order to enhance the instrument performance and reduce the 39 fluorescence and other interferences: (1) The photo- multiplier tube, is moved farther away from the sample cell excitation flash is provided with an extension tube. The distance between the sample and.the:detector is increased from 4 cm to 40 cm. The probe beam is focused at the detector instead of at the sample and a slit is placed near the detector to limit the solid angle of fluorescence from the sample. This modification reduces the fluorescence interferences and excitation laser flash artifactual effects tremendously, and makes the instrument capable of detecting IV680 decay in the near infrared region where the sample has very strong fluorescence [3]. (2) The probe beam is defocused at the sample, which reduces the intensity of the probe beam on the sample so that photo-excitation of the sample is not a serious problem. The electronic shutter that was designed to protect the sample from the probe beam is no longer needed. (3) The sample is oriented at about 45 degree to the actinic flash in order to obtain more effective and more uniform excitation. The modified optical subsystem layout is shown in Figure 2-2. All components are mounted on an optical rail bolted to a heavy length of colorlithe, secured to a laboratory table top. This helps to suppress the noise from environment. The probe beam light source is provided by a 75 W Xenon Arc Lamp from Photon Technology with a LSP-200 universal power he :8 nee pho pro It k and grat. with givin reSPEC 40 Filter Filler Sample Slit . I Arc Monochromater 5‘”! >~r PMT Lamp Extenllon tube Lens /\ T l Laser Flash Figure 2-2 Optical layout of the transient absorption spectrometer used in the studies of the electron transfer in PSII (only relative positions are shown). supply. Though the arc lamp is not as good as a tungsten- halogen lamp in term of signal/noise level, its spectral radiation covers the visible as well as the ultraviolet and near infrared regions, which makes it convenient for studying photosystem II. 'The wavelength selection of the probe beam is provided by an H-20 UV-VIS monochromator from Instruments SA. It has two ion-etched concave holographic gratings for the UV and the near-IR, as well as a normal concave holographic grating for the UV-VIS region. The monochromator is equipped with fixed interchangeable slits of 0.5, 1.0, and 2.0 mm, giving an optical bandwidth of 2.0, 4.0, and 8.0 nm respectively, which are suitable for transient absorption studies. 41 The sample compartment consists of a filter holder, sample holder, and a 50 mm diameter silica concave lens with a focal length of 10 cm. 'The optical filter before the sample is used to prevent second order diffraction from the monochromator from reaching the sample. The filters after the sample are used to cut off the excitation laser flash and fluorescence from the sample which allowing the probe beam to pass through. The sample holder is modified in a such way that the sample can be oriented at any angle between 0 and 90 degree, which makes it possible to optimize the cross section for both probe beam and excitation flash. The long focus UV lens is used to reduce the intensity of the probe beam on the sample and to focus the probe beam farther away from the sample so that the detector can be mounted at a distance of 50 cm from the sample. This arrangement reduces the solid angle of fluorescence from the sample tremendously and makes the interference of fluorescence from the sample insignificant when measurements are made at 820 nm. A Nd:YAG pulsed laser from Quantum-Ray is used as the excitation source. The 10 ns excitation pulse at 532 nm is generated by frequency doubling the 1064 nm laser radiation in a KDP crystal. The 355 nm pulse can be generated by frequency tripling. The 355 nm exciting pulse is necessary when 'measurements are made around 550 nm region to monitor the C550 band shift in PSII. th ral on det dis1 phOtc sens] Stf‘on, dekgl 42 Detecting and Signal Processing The detector used in the instrument is a 50 mm diameter photomultiplier tube, 9659QB from EMI. It has 11 venetian blind dynodes and an extended S-20 window. The signal from the PMT is first sent to the pre-amplifier that is located behind the PMT housing. The amplified signal is then sent to the fourth order low pass active filter to filter out high frequency noise and the filtered signal is input to the sample/hold amplifier for further amplification and DC level subtraction. Finally the processed signal is sent to the analog to digital converter for digital processing and data storage. The block diagram for the detecting and signal processing subsystem is shown in Figure 2-3. The signal to be detected is the change of absorbance of the sample following the actinic flash. It is usually in the range of 0.01% to 0.1%. Because the signal to be detected is on top of a strong background, special considerations of the detecting system must be made so that the signal is not distorted. The detector used in the instrument is an 11 dynode photomultiplier tube, which has very high gain and sensitivity. Since the signal to be detected comes with a strong background, the PMT can be easily saturated by background radiation. In< UJ "5 I T ' I T l i l . l C) 200 400 600 800 1000 MICROSECOND Figure 3-2 Microsecond time courses of the absorption changes at 325 nm (trace A) and 820 nm (trace B) with tris- washed.PSII. [Chl]=215.7 ug/ml, optical.path=0.14 cm, excita- tion flash repetition rate 5.8 Hz. 2.5 mM Fe(CN)6'3/Fe(CN)6'2 were added as the electron acceptor system. The transient in the first few us was unreliable because of flash artifacts. 79 I2- I 2 E 8‘ * E L) - \ ‘— I V 4_ in A (UNTREATED) Z I WWI/[iii O B .4 W311 WV‘IWW with \l ' _ Z “M .Ji‘HVW'wV-l’v. t: o- B (NHZOH) * >< LLJ “4 I I I _T 7 I I I . I O 100 200 300 40C) 500 MILLISECOND Figure 3-3 Millisecond time courses of the absorption changes at 325 nm with tris-washed PSII (trace A) and NHZOH treated tris-PSII (trace B). [Chl]=215.3 [lg/ml, optical path=0.14 cm, the time between the excitation flashes was 1.98 second. 2.5 mM Fe(CN),,'3/Fe(CN),’°2 were added as the electron acceptor system. The transient in the first few us was unreliable because of flash artifacts. re re ti ir. PC of be es in She re; 80 determined by the rate of the charge recombination. Figure 3-2 and Figure 3-3 shows the kinetic traces of the absorption changes at 820 nm.and 325 nm.in the microsecond and in the millisecond time scales, which represent QA' and P‘680 decays, respectively. The P"680 (trace B in Figure 3-2) in the microsecond scan shows monophasic decay with a halftime of 226 i 27 us, whereas the Q‘ (trace A) shows a biphasic decay with halftimes of 223 i 11 us and 45 i 1 ms. The amplitudes of both P’680 and QA' depend strongly on the flash repetition rate, this is shown in Figure 3-4 (trace A) and Figure 3-5. At low flash repetition rate, the P‘680 amplitude is small on the tens of microseconds time scale (Figure 3-4, trace A), and Q{'decays mainly in the millisecond time range. IMore than 80% of the QA' decays in the millisecond time scale at a flash repetition rate of 1.2 Hz. ‘Upon increasing the flash repetition rate, PV680 becomes observable in the microsecond time range, and at the same time, the microsecond Q; phase is increased at the expense of its millisecond phase decay. Following Haveman and Mathis [54] , we attribute the appearance of Q;]T”680 recombination to those centers in which Yf'has not been reduced in the time between flashes. From Figure 3-4, we estimate that the halftime for Yf'reduction by ferrocyanide in tris-washed PSII is approximately 300 ms. lThis reaction is about 40 times faster than is observed for the Yf/ferrocyanide reaction in tris-washed chloroplasts [55], which may reflect 81 5_ .l E 4- B (NH2OH TREATED) E 9e . 2 L) \\\ 3—~,£ // Z / Q 2—~ B ' A (UNTREATED) Z - ]_. X 1 _ LIJ O _. /. O F I T I I r T I I I l 1 O 2 4 6 8 IO 12 FLASH RATE (Hz) Figure 3-4 The dependence of the absorption changes at 820 nm on the flash repetition rate. Trace A is tris-washed PSII, trace B is NHZOH treated tris-PSII. 82 8-— . Lg ////. A WW” E3 670 . .X. E L) . \ v_ O \_/ 4‘— // 8 / E _ L) ZZ E 2— >< DJ 0 I I I I I I I I I I I I O 2 4 6 8 IO 12 FLASH RATE (HZ) Figure 3-5 The dependence of the absorption changes at 325 nm on the flash repetition rate in tris-washed PSII. 83 the greatly reduced diffusion barrier to the external reductants in the resolved PSII preparation. The extinction coefficient for P680 at. 820 nm has not been defined precisely, the reported value has a range of 4.2 to 7.0 mM'1cm'1 [11,23]. The measurements we performed at 325 nm and 820 nm allowed us to calibrate the extinction coefficient of P680 at 820 nm against the better defined value of QA. Knowing the percentage of the QA' that undergoes recombination with P’680, 63% at excitation repetition rate of 5.8 Hz, and the extinction coefficient of QA, 13.0 mM'1cm’1 at 325 nm, we obtained an extinction coefficient for P680 at 820 nm of 6.6 i- 0.3 mM'1cm’1. QA'/P*680 Recombination in NaCl Washed PSII Without addition of Ca”, NaCl washed PSII membrane fragments are unable to carry out the normal electron transfer reactions that result in oxygen evolution [52,53]. Under repetitive excitations, electron transfer on the donor side of PSII in NaCl washed PSII is retarded and charge recombination between QA' and P’680 occurs. Figure 3-6 shows the microsecond decay traces of QA' and P‘680 in NaCl washed PSII. The decay is similar to that in tris-washed PSII except that it is somewhat faster. By fitting the data, we obtained decay halftimes of 125 t 6 us for QA' (fast phase), and 138 i 9 us for 13*680 . lC‘EIqt 84 '15- A E E 10— * 2 ~. ~m Q ~ w \ “A :2 5.. Z A O W I5 " f WW “IQ/“WWW /V Z VWVfVVl/Vli_v/\V,AMWIWW ; “WW B LLJ W I T “5 ‘ l ‘ i ‘ I ‘ I ’ l O 200 400 600 800 1000 MICROSECOND Figure 3-6 Microsecond time courses of the absorption changes at 325 nm (trace A) and 820 nm (trace B) in NaCl washed PSII. [Chl]=214.1 ug/ml, optical path=0.14 cm, excitation flash repetition rate 5.8 Hz. 2.5 mM Fe(CN)6"3/Fe(CN)6"2 were added as the electron acceptor system. The transient in the first few us was unreliable because of flash artifacts. 85 Q"/P*680 Recombination in NHZOH Treated Tris-PSII NHZOH treatments of PSII have been reported to inhibit water oxidation and electron transfer from Yz to P+680 [19,33,34]. When electron transfer in PSII is inhibited, the decay of the charge separated state QA'/P"680 occurs primarily by charge recombination. The absorption changes following the excitation with NHZOH treated tris-PSII are shown in Figure 3-7 and Figure 3-3 (trace B). The P‘680 decay was fit to a halftime of 236 i 24 ps. QA' decays in a biphasic manner with halftimes of 240 i 12 us and 80 1' 2 ms. Compared to recombination in tris-washed PSII, NHZOH treated PSII show a similar fast rate, but the ratio of the microsecond phase Q{ decay is higher. The extinction coefficient for P680 at 820 nm in NHZOH treated tris-PSII, 7.2 i- 0.3 mM'1cm“, is slight higher than the value in tris-washed PSII. With NH20H treated PSII, high flash repetition rate are no longer required in order to observe the charge recombination reaction. This is shown in Figure 3-8 and Figure 3-4 (trace B). The decays of P’680 and QA' are almost independent of the flash repetition rate. The high repetition rate is not needed since the electron donation from Yz to P‘680 is blocked by NHZOH treatment. The EPR experiments performed by Hoganson et a1. [35] and Ghanotakis et al. [33] showed that the YZ is unable to be oxidized by P‘680 under the treatment of NHZOH. 86 1 5 - S , E l O .X. E Q -I \ ' V 'V\\ C) ‘ TWP» 5 ] LMW NW [WWI F— __ ,. X 0 DJ — 5 . I . I «r— T “"T O 200 400 600 800 1000 MICROSECOND Figure 3-7 Microsecond time courses of the absorption changes at 325 nm (trace A) and 820 nm (trace B) with NHZOH treated tris-PSII. Experiment conditions were same as those in Figure 3-2. The transient in the first few us was unreliable because of flash artifacts. 87 8T - FAST PHASE A O E E 5‘ .x. :E c Q _ \ V 4_ i5 E— l L) Z o O F— 2~ o SLOW pHASE if] e o O I I I I I I I I I I I I O 2 4 6 8 IO 12 FLASH RATE (Hz) Figure 3-8 The dependence of the absorption changes at 325 nm.on the repetition rate of the excitation flash in NHHMI treated tris-PSII. 88 Light is required for NHZOH to react with the donor side of PSII to disable the electron transfer capability of Y,. Consistent with.this earlier observation, it was also observed in our experiments (data not show) that without illumination after the addition of NHZOH, the decay trace of the first 20 scans is different from that of the second 20 scans. After about 50 flashes these differences become unnoticed. The experiments we performed here used 60 to 80 flashes to meet the light requirement of the NHZOH inhibition before the measurements were taken. Using pre-flashes instead of pre- illumination for NHZOH inhibition treatment provided a better way to control the inhibition process and avoid non-uniform illumination and heat damage to the samples. c-sso Bandshift In photosystem II, QA'/QA and Qg/Qalhave very similar UV absorption difference spectra. Thus, it is impossible to distinguish the Q{ contributions from that of Q{ when measurements are made only at 325 nm. However, the reduction of QA induces an absorption band shift of the pheophytin a molecule centered at 545 nm [44-46] . The charge on 98' on the other hand, has very little effect on this shift. Thus, the absorption difference at 540 nm minus that at 550 nm has been often used as a measure of the amplitude of C550, a local electrochromic indicator of the concentration of Q; [16,44]. 89 MW lAe=l£> WWII/Wm EXTINCTION (l/CIVHmM) “AA T I l T j l O 2CK) I I I 600 800 1000 MICROSECOND I 4CM) Figure 3-9 Microsecond time courses of the C550 band shift in tris-washed PSII (trace A) and NHZOH treated tris-PSII (trace B). [Chl]=315.7 pg/ml, 2.5 mM Fe(CN),;3/Fe(CN),;2 were added as the electron acceptor system. 90 Table Halftimes of the Microsecond Decays Treatment EV680 (us) Q{ (us) C550 (us) Tris 223 i 11 226 i 27 225 i 29 NaCl 125 i 6 138 i' 9 NHffli 240 i 12 236 i 24 233 i 29 Measurements of the C550 band shift were performed with tris-washed PSII and. THEOH treated tris-PSII membrane preparations. Figure 3-9 shows the time courses of the 0550 band shift measured as the absorption difference at 540 nm minus that at 550 nm. The decays are biphasic, with the fast phase in the microsecond and.the sl w phase in the millisecond range. The halftimes of the faster phase are 225 i 29 us for tris-washed PSII and 233 i- 29 us for NHZOH treated tris-PSII. The ratio of the microsecond phase of C550 decay is about 70%. These results are in good agreement with the measurements made at 325 nm, and imply that the contribution from Q; is small. The differential extinction coefficient for the 540 nm minus 91 550 nm obtained in our experiment was 2.1 qucm”, this is close to the reported value of 2.2 mM'1cm'1 [46]. Piscussion The results we obtained here show that the reaction we observed is the charge recombination reaction from the charge separated state Q{/P”680. This contention is strongly supported by the decay kinetics of Q; and.P”680, which show near identical decay halftimes. The charge recombination process depends on the different inhibition treatments and experimental conditions. Both decay pathways and rates can be modified by using different inhibitors or changing conditions. In tris-washed PSII, the electron donation by Y, to the reaction center P"680 is still active. This is demonstrated in our experiments by the fact that at low excitation rates, almost no P‘680 can be detected in the one hundred microsecond time scale. The rate limiting step of the electron transfer on the donor side of tris-washed PSII is the reduction of the oxidized secondary' donor Yf. From “the results of the dependence of the absorption changes of [”680 on the excitation repetition rate we estimated that the halftime of Y; reduction in tris-washed PSII in the presence of 2.5 mM ferri/ferrocyanide at pH 6.0 is about 300 ms. Our data do not show any direct involvement of Q8 during the charge recombination process. In intact PSII membrane be tr re ex th of Che the tra ban to com; P‘68 reco Syne FOSS. samp; Site. retar Charg. by th. Change 92 fragments or chloroplasts, the QA'to QB electron transfer has been shown to take place in about 200 us [16,47]. If this is also the case in tris-washed PSII, the, QA' to QB electron transfer should compete effectively with the charge recombination process; however, this is not observed in our experiments. Our results show a very similar kinetic trace of the absorption changes detected at 325 nm and the time course of the C550 band shift that is caused mainly by the negative charge on 0,. This rules out any significant contribution of the electron transfer from QA' to Q8, since this electron transfer reaction will change the decay kinetics of the 0550 band shift dramatically. It is not clear why the electron transfer reaction of Q{ to Q3, though it has a comparable rate constant, can not compete with the charge recombination reaction between Q{ and IV680. Very recently Gerken et a1. [18] measured the charge recombination reactions between QA' and P’680 in cyanobacterium Synechococcus sp. and. obtained similar results. It is possible that plastoquinone is severely depleted during the sample preparation, which will effectively deplete the QB site. However, another possible reason for the dramatic retardation of the QA' to QB electron transfer is the positive charge on the reaction center'PV680. The Coulomb attraction by the positively charged reaction center I”680 can induce changes of the free energies of nearby reactions, which 93 Ferri/ Ferrocyanide ps 8 Figure 3-10 The scheme of the electron transfer pathways in tris-washed PSII. according to Marcus electron transfer theory [48], will alter the rates of electron transfer. The different behavior of the dependence of the charge recombination reaction on the repetition rate of the excitation flash with NHZOH treated PSII implies that a different inhibition mechanism is in place. In tris-washed PSII, the inhibition is achieved by removing electron donors to Yf, while the electron transfer from Y2 to P‘680 is not 94 affected greatly. However, in NHZOH treated tris-PSII, the electron transfer from YZ to P‘680 is greatly retarded or blocked and the electron donation of Y2, to P‘680 no longer competes with the QA'/P’680 charge recombination reaction. At least two inhibitory actions of NHZOH treatment have been reported [19,33]. In one case it acts as an electron donor to PSII, possibly competing with water, and in the other case, it inactivates the donor side of PSII, retarding the electron transfer from Yz to P‘680. Our results clearly favor the second of these two models. Hoganson and Babcock [35] has suggested that the NHZOH inhibition may involve a reversible oxime formation at one of the carbonyl groups of P680, which might lower the redox potential of P‘680 and prevent it from oxidizing Y2. Our results suggest that the NHZOH inhibition site is more likely localized near YZ rather than at the reaction center P680. This will be discussed in more detail in chapter IV. The decay of the state QA’/P*680 via the charge recombination reaction has been studied by using both optical and EPR techniques; the halftimes obtained range from 100 to 800 us [11,18,19,22,28-31,35] . Recently, Gerken and coworkers [18] observed that three exponential decay phases with half times of 170 us, 800 us and 6 ms are involved in the charge recombination reactions in cyanobacterium PSII membrane preparations, and claimed that these multiphasic kinetics were 95 due to a distribution of different structural states of the reaction center polypeptides. Our results with tris-washed PSII from spinach do not resolve significant multiphasic kinetics of the charge recombination reaction. Instead a single exponential decay with.a halftime of 223 us is observed in the microsecond range. The charge recombination reactions in our preparations are most likely taking place from the same structural states of the reaction center polypeptides, since heterogeneity in these states can result in a non-exponential behavior of the reactions [49]. Such behavior is not observed in our experiments. THMOH treatment of tris-PSII does not change the rate of the charge recombination process, which suggests that the valence of‘Kzis not critical in influencing the kinetics of the reaction. In NaCl washed PSII, the charge recombinatiOn reaction of Q;]P”680 is faster, with a halftime of about 130 ps. Unlike tris washed-PSII, NaCl washing removes only the 17 KDa and 23 KDa polypeptides, the 33 KDa polypeptide and the manganese ions associated with the water splitting complex are retained [31,41]. The NaCl washed PSII membrane preparations is able to 'undergo several turnovers before the normal electron transfer is inhibited [41]. Moreover, oxygen evolution activity can be restored up to 75% upon the addition of Ca2+ [52,53]. However, in tris-washed PSII membrane preparations, all three extrinsic polypeptides, as well as the manganese 96 ions, are released, and the redox properties of Y2 are modified. These differences in the micro-environment at the donor side of PSII influence the rate of the charge recombination reaction. The positively charged species at the donor side of PSII may exert Coulomb effects and result in a faster charge recombination reaction. The changes of the redox potential of Y2 under tris-washing treatment may also play a role in the recombination rate. The mutagenesis experiment carried out by Metz and coworkers [50] shows that when Yz is replaced by phenylalanine, a residue with higher redox potential, the charge recombination reaction between QA' and P‘680 is slowed down to 1 ms. Coaglusion The time evolution of the charge separated state QA'/P"680 under different treatments can be used to obtain valuable information of the micro-environment of PSII. The charge recombination of the state QA'/P*680 can be detected optically by measuring the time courses of the absorption changes at 325 nm (QA') and 820 nm (P’680) . Our results show that charge recombination occurs in tris-washed PSII at high repetition rates of the excitation flash. NHZOH treatment removes the requirement of high flash repetition rate. This difference comes from the different inhibitory states. Tris washing inhibits the re-reduction of Yz“ by its physiological donor 97 whereas the electron transfer from Yz to P‘680 is not blocked. NHZOH treatment greatly retards the electron transfer from Yz to P‘680 and keeps Yz in its reduced state. The rate of the charge recombination reaction in NaCl washed PSII is faster than that in tris and NHZOH treated PSII. 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DekI CHAPTER IV CHARACTERIZATION OF P‘GSO/PGSO AND YZ+IYZ ABSORPTION DIFFERENCE SPECTRUM mam Photosynthesis in higher plants involves a series of electron transfer reactions that lead to water oxidation and (X5 reduction. These electron transfer reactions are carried out by individual electron carriers that undergo reduction or oxidation during the electron transfer process. The absorption changes associated with the redox activity of these electron carriers can be detected by transient absorption spectroscopy. The wavelength dependence of the absorption changes, i.e. the absorption difference spectra, can be used to identify and characterize these electron.carriers and their micro-environments. Absorption difference spectra have been widely used to characterize the electron carriers in PSII [1-4]. The first full absorption spectrum of QA-IQA was obtained by Van Gorkom [5] and was identified as a plastoquinone molecule due to its similarities to the in vitro spectrum of plastoquinone. Dekker and coworkers in 1984 [3] reported the detailed 103 p1 ab its a F abs HOWe P01 y] tyros Simil Obtaii I31. [1 Y2 was 104 absorption difference spectrum of QA'/QA and Y2”/Yz in tris- washed PSII in the presence of ferricyanide and 3-(3',4'- dichlorophenyl)-1,1-dimethylurea (DCMU) by analysis of the flash induced kinetics of millisecond absorption changes, prompt and delayed fluorescence, and EPR signal 11,. It was shown that the absorption difference spectrum of QM'lQA consists of two negative peaks at 268 nm and 433 nm, and four positive peaks at 325, 398, 416, and 449 nm, The spectrum of Yf/Yz consists mainly of positive bands at 260 nm, 300 nm, and 390-450 nm on which a chlorophyll band shift around 438 nm is superimposed. Similar results were obtained with native preparations [6]. The chemical identification of the donor Yz by its absorption difference spectrum was not successful because of its low spectral resolution. It was originally aSsigned to be a plastoquinone molecule [3,7] due to the similarity of its absorption difference spectrum in the ultraviolet range. However, recent mutagenesis experiments [8-11] have shown that Yz is a tyrosine residue located at position 161 of the D1 polypeptide. The in vitro absorption difference spectrum of tyrosine radical [12,13], Ter'lTerH, has features that are similar to those of the Yf/Yz absorption difference spectrum obtained by Dekker et al. [3] and more recently by Gerken et a1. [14] in the UV range. The original misassignment of the Yz was due to the low resolution of the absorption difference in re wh. dii abs et Syn: diff arou. and e afoun (7218‘) 105 spectrum as well as to the unresolved EPR spectrum of Y; [IS-l7]. The absorption difference spectrum of P*680/P680 in the UV range has not been studied as extensively as that of Q“'/QA and Yz’le. The reasons for this are that the decay of P‘680 is fast (in the nanosecond time scale) and that there are difficulties in separating contributions from other components. Weiss and Renger [18] measured the P‘680/P680 absorption difference spectrum from 250 nm to 400 nm in trypsinized and NHZOH treated PSII membranes. They found that there were differences in the P‘680/P680 absorption difference spectrum near 300 nm and 360 nm in trypsinized and NHZOH treated PSII membranes. However, the spectra were not well resolved and the measurements were not performed above 400 nm where the most characteristic features of the P’680/P680 difference spectrum are located. A more detailed P‘680/P680 absorption difference spectrum was obtained recently by Gerken et al. [1] in tris-washed PSII from cyanobacterium Synechococcus sp. . They found that the P‘680/P680 absorption difference spectrum is dominated by a very sharp bleaching around 434 nm, by absorption increases around 305, 345, 400 nm and above 450 nm, and absorption decreases below 290 nm and around 375 nm. A comparison with the in vitro spectrum of Chla’/Chla in CHZCl2 [19] reveals similarities in the wavelength range over which absorption changes occur, although 106 significant differences are apparent. These differences might be caused by differences in aggregation state as well as in the local environment. The absorption difference spectrum of P‘680/P680 and Yf/Yz can be obtained by various methods. However, the most important aspect to be considered is how to separate the contributions from other components that are active during the electron transfer reactions. An inaccurate subtraction of those interferences can result in distorted spectrum. In the experiments described here we obtained the absorption difference spectrum of P‘680/P680 and Yz"/YZ by monitoring the charge recombination reactions of the state Q{]P”680 in tris and NHZOH treated PSII. The advantages of this method are the following: (1) It involves fewer electron transfer reactions so that the different pathways can be defined more accurately; (2) The different electron transfer reactions can be relatively easily separated since they occur on.different time scales (e.g. , QA'/P‘680 recombination occurs in the microsecond time scale, while Q{/Y{ recombination occurs in the millisecond time scale). The NHZOH treatment that inhibits the electron transfer from Yz to P’680 is not well understood. Whether the inhibition takes place at the reaction center P680, at the Yz site, or remotely in the protein is still an open question. In the experiments described here we have performed 'ku.:... . mike—f ii th tr tel car for init take 1491211): 200 L light 0f tr Chapte 107 measurements in NHZOH treated tris-PSII in order to determine how NHZOH treatment affects the difference spectrum. The modifications of the absorption difference spectra of P’680/P680 and of Yf/Yz with NHZOH treated samples provides us insight into the mode of NHZOH inhibition. Materials and Methods The experiments were performed with tris-washed photosystem II membranes. The PSII membranes were isolated from spinach according to the method described by Berthold et al. [20] with some modifications [21]. The preparation procedures were described in detail in chapter III. Tris inactivation of PSII membranes was performed by incubation of the PSII membranes in a solution containing 0.8 M tris(hydroxymethyl)aminomethane and 0.5 mM ethylenediamine tetraacetic acid (EDTA) at pH 8.0. The incubation process was carried out at a chlorophyll concentration of 0.5 mg/ml at 0°C for 20 minutes under room light. The NHZOH treatment was initiated about 5 to 10 minutes before the measurements were taken. 2.0 mM NHZOH was added to the tris-washed PSII membrane preparations at a chlorophyll concentration of about 200 ug/ml. 60-80 pre-laser flashes were given to meet the light requirement of NHZOH inactivation [18,22,23]. Details of the tris and NHZOH treatments were described in the chapter III. oi th we di: to flu use. SCaI prng VEre 108 The optical measurements were performed with a chlorophyll concentration around 200 ug/ml in.a buffer consisting of 0.4 M Sucrose, 50.0 mM MES, 15.0 mM NaCl, and_pH 6.0. Lower Chl concentration, around 100 ug/ml, was used in the 250-280 nm range because of the strong sample absorption in that region. The experimentSIwere carried out with.the transient.absorption spectrometer described in the chapter II. The sample cell used was a 0.1 cm optical path quartz cuvette that gave an effective optical path length 0.14 cm when oriented at 45 degree relative to the probe beam. The slit used with the monochromator were 1.0 mm that gave a spectral resolution of 4.0 nm. Three glass filters ( U330 for the wavelength range of 250-280 nm, U340 for the range of 280-370 nm, and B390 for the range of 370-480 nm; the filters were from Hoya Corp.) were used, one behind the monochromator to block the secondary diffraction from the monochromator and two in front of the PMT to protect the PMT from the excitation laser flash and fluorescence from the samples. The instrument time constants used were 5 us for microsecond scans and 1 ms for millisecond scans. The excitation flash was provided by a Quanta Ray Nd:YAD laser. The wavelength of the excitation flash was 532 nm. All measurements were performed at room temperature. The data were analyzed by using the kinetic fitting program KFIT from OLSI. The absorption difference spectrum were constructed by plotting the amplitudes of the absorption e: se wi ThI unc‘ equ About exCit 109 changes of corresponding phases against the wavelength. The extinction coefficients were calculated under the assumption of 250 Chl molecules per reaction center- Resuits P’GSO/PGSO Difference Spectrum The charge separated state Qp/PV680, which is created in about 300 ps following the visible excitation [24-27], decays in the microsecond time scale when high repetition rate flash excitation was applied to tris-washed PSII. At 5.8 Hz flash repetition rate, about 63% of the Q{/P”680 is still present at 5 us (the time constant used in the experiments) following the excitation flash (Figure 3-4 in the chapter III). The charge separated state decays by recombination between QA' and P’680 with a halftime of 230 ps (Figure 3-2 in the chapter III). Thus, the electron transfer reactions in tris-washed PSII under the above conditions can be represented by the following equation: t55ps 0.37 YZ + 1.0 P680 + 1.0 QA-———————> 0.37 Y; + 0.63 P”680 + 1.0 QA° About 37% of Y2 is still in reduced state prior to the excitation flash and results in a 63% detectable P"680 in the tr no the 1‘ 981 Spec We; to [”1 State 110 microsecond time scale. The initial amplitudes of the absorption changes after 5 us (the instrument response time) following the excitation flash are plotted against wavelength in Figure 4-1. The 230 ps phase of the QA'/P*680 decay arises from the charge recombination reaction between QA' and P‘680, a contention that is strongly supported by the observations that Q,” and P‘680 have near identical decay halftimes. Thus, the absorption difference spectrum of the 230 ps phase as shown in Figure 4-2 should represent the total absorption difference spectrum of QA'P*680/QAP680. The P‘680/P680 absorption difference spectrum can be obtained by subtracting the QA'/QA contributions from the QA'P”680/QAP680 difference spectrum. Figure 4-3 shows the P’680/P680 difference spectrum in tris-washed PSII. The subtraction was carried out by normalizing the spectrum at 325 nm under the assumption that the absorption change at 325 nm is due to QA' only [3]. The QA'lQA absorption difference spectrum was taken from the results of Dekker et. al. [3]. The absorption difference spectrum of Chlorophyll a’lChlorophyll a in CHZC12 [19] is overlayed with the P‘680/P680 spectrum in Figure 4-3 for comparison. In NHZOH treated tris-PSII, the electron transfer from Yz to P‘680 is inhibited [22,28,29] and the charge separated state QA'/P*680 decays mainly via the charge recombination 111 15—— - IO—~ :E l E 5—— a * J \ \8’- '2' . / \ I” Q \ O X Z O -5‘ C H % ;—10~ >< . DJ __15_l ‘20 IIIIEIIIIIIIIIIIIIIIIIIII 250 300 350 400 450 500 WAVELENGTH (NM) Figure 4-1 Absorption difference spectrum of the initial amplitudes of the total absorption changes after 5 us following the flash excitation in tris-washed PSII. [Chl]=215 ug/ml, optical path=0.14 cm, excitation flash repetition rate 5.8 Hz. 2.5 mM Fe(CN);3/Fe(CN)g‘ were added as the electron acceptor system. 112 /'\ 5_ ‘ :5 Xxx E fix '2 \ I o 0 . -.. > f X j j V - / Z V 9 J. }_ s L) Z - l..— >< LJJ —IO— —15 250 1' 300 I} 356 " A00 IT 450 I] 500 WAVELENGTH (NM) Figure 4-2 Absorption difference spectrum of the 230 us decay phase in tris-washed PSII. 113 20— TO- A EXHNCTKWJ(I/CM*HAM | I Ei E3 1 1 —30- \ (I 7 l! \I \I —4OIiIrifilljri—[Tjrfl’lllIlllllli 250 300 350 400 450 500 WAVELENGTH UNM) Figure 4'3 Absorption difference spectrum of the reaction center P’680/P680 in tris-washed PSII (open circle, continuous line) and the spectrum of Chla”/Chla in CHZCl2 (dash line). 114 reaction between QA' and P’680. Under the conditions of 2.0 mM NHZOH, 60 pre-flashes, and 5.8 Hz flash repetition rate, NHZOH treated tris-PSII shows that about 79% of the state QA'/P*680 is still present after 5 us following the excitation flash (Figure 3-8 in the chapter III). The fraction of reaction centers, 21%, that are reduced in the sub-microsecond time scale might be the result of incomplete inhibition by NHZOH treatment, or reflect reduction by electron donors other than Y2 [30]. The electron transfer reactions of the state QA'/P*680 after 5 us following the excitation flash in the NHZOH treated tris-PSII can be represented by the following equation: tssus 0.21 donor + 1.0 P680 + 1.0 QA ———> 0.21 donor (oxidized) + 0.79 P‘680 + 1.0 QA' Reaction centers, not reduced in the sub-microsecond time scale, undergo charge recombination between QA’ and P‘680 with a halftime of 230 ps. Thus, the absorption difference spectrum of the 230 us phase of the QA'/P*680 decay represents the total spectrum of QA'P*680/QAP680 in NHZOH treated tris- PSII. Subtracting the QA'/QA contributions from this total spectrum, we obtained the absorption difference spectrum of P‘680/P680 in NHZOH treated tris-PSII (Figure 4-4 open triangle). The P‘680/P680 spectrum in tris-washed PSII is A.A.. A l l 115 20- /—\ 1 I E O E q . 'X' '2 A A . O O A- a4 88an \ :3!) a 5 Z 9 -IO- A] t— L) E . t__ X LI_J —20- Z30 I T I I I l I l T r T I I l 1 71 l l I ] T l I I I ’ 250 300 350 400 450 500 WAVELENGTH (NM) Figure 4-4 Absorption difference spectrum of the reaction center I”680/P680 in tris-washed PSII (open circle) and in NHZOH treated tris-PSII (open triangle). The NHZOH treatment was performed with the addition of 2.0 mM NHZOH and 60 pre- flashes before the measurements were taken in tris-washed PSII. The experiment conditions were the same as in Figure 4-1. de we Spe Y,‘/ COD' subt Spec Carr,- diffs: and Ha 116 also presented in the Figure 4-4 as a comparison. Yf/Yz Difference Spectrum In tris-washed. PSII, the :millisecond. decay phase is dominated by the recombination reaction between QA' and Yz*. In order to increase the amplitude of the millisecond decay phase signal, the time between flashes was extended to 1.98 seconds. Under this condition, about 71% q; decays in the millisecond time scale with.a halftime of 45 ms (Figure 3-5 in the chapter III). This 45 ms decay phase presumably arises from.the charge recombination reaction between Q{ and Y;'[3]. EPR data on the decay of Y; are also consistent with this assumption [3,31]. The absorption difference spectrum of the millisecond decay phase of the charge separated state Q{/P”680 in tris- washed PSII is shown in Figure 4-5 (open circle). This spectrum represents the Q‘Yf/QQQ difference spectrum. The Yf/Yz spectrum can be obtained by subtracting the QA’/QA contributions from this spectrum. Figure 4-6 shows the subtracted spectrum that represents the absorption difference spectrum of YfYY, in tris-washed PSII. The subtraction was carried out under the assumption that the absorption changes at 325 nm are due to Qg'only [3]. The in vitro absorption different spectrum of tyrosine taken from the results of Bent and Hayon [12] is overlayed in the Figure (dashed line). The t1 ft in spe sang of t afte. Spec! comp] CORtr; Chlorc SPECtr Contril 117 in vitro tyrosine spectrum in Figure 4-6 is attributed to the formation of the phenoxy radical TryO' resulting from the loss of the phenolic hydrogen from tyrosine in water. In NHZOH treated tris-PSII, the amplitude of the millisecond decay phase is very small even at very low flash repetition rate (Figure 4-5, open triangLe). IUQOH treatment inhibits electron transfer from Y2 to P’680 and results in the decay of the charge separated state Q{]P”680 mainly via the charge recombination reaction between Q,’ and P‘680. Under the same conditions as the untreated tris-washed PSII, NHZOH treated tris-PSII shows that only 29% Q{ is present at 1 ms following the excitation flash. This fraction of Q{ decays in the millisecond time scale with a halftime of about 80 ms (Figure 3-3 in the chapter III). The same procedure used to obtain the Yf/Yz difference spectrum in tris-washed PSII was employed for NHZOH treated samples. Figure 4-7 shows the absorption difference spectrum of the 80 millisecond decay phase in NHZOH treated tris-PSII after subtracting the QA'/QA contributions from the total spectrum in 4-5 (open triangle). The spectrum shows very complicated features. In the visible region, the contributions may arise from the oxidized reaction center chlorophyll P‘680. The complicated absorption difference spectrum in NHZOH treated tris-PSII might be the result of the contributions of several electron transfer components. elm.lt. 118 8.. J E 4—~ ,, E IX . E: _ ff, fr > Qty/kl #3:“: V O , ””qu g A... W I: - L) E :2 4 DJ “8 I II I I II I I II I I II I I I II I I I rI 250 300 350 400 450 500 ‘WAVELENGTH HVV) Figure 4-5 Absorption difference spectrum of the millisecond decay phase in tris-washed PSII (open circle) and in NHZOH treated tris-PSII (open triangle). [Chl]=215 ug/ml, the time between excitation flashes was 1.98 second. 2.5 mM Fe(CN){3/Fe(CN)g* were added as the electron acceptor system. Fig tri QI/ Clrc abSc Wate 119 EXWNCTKNJ(I/CM*WWD _2 IIIIIT7IT1T I YT] [Till I [FT] 250 300 350 400 450 500 \NAVELENGTH (MID Figure 4-6 Absorption difference spectrum of the Elf/Yz in tris-washed PSII. The spectrum is obtained by subtracting the QA'IQA contributions from the spectrum in Figure 4-5 (open circle). The overlayed spectrum (dashed line) is the absorption difference spectrum of the phenoxy radical Ter' in water. ... “A.A.-.... ‘5 .‘ 120 15— g 10m f E -« * E U r \ 5 z “ Wm Q o i A [\L [V I~~ V ‘6 L) E; _ }__ >< DJ —5—« ‘10IIIIIIIIIIIIIIIIIIIIIIIII 250 300 350 400 450 500 WAVELENGTH (NM) Figure 4-7 Absorption difference spectrum of the millisecond decay phase in NHZOH treated tris-PSII after subtracting QA'/QA spectrum from the spectrum in Figure 4-5 (open triangle). CC SP sut spe 121 Discussions Monitoring the decay of the charge separated state QA'/P*680 provides a direct way to determine the P‘680/P680 absorption difference spectrum. This method uses tris inhibition to extend the lifetime of 3’680 into the microsecond range so that the charge recombination reaction between QA' and P‘680 can be observed. The advantages of the method are that the decay pathway is relatively simple, fewer electron transfer reactions are involved, and the various components involved can be relatively easily separated spectrally and kinetically. The P’680/P680 absorption difference spectrum obtained by subtracting QA'lQA contributions from the QA’P+680/QAP680 spectrum is shown in Figure 4-3. The spectrum has positive bands around 310, 345, 400 and 470 nm, and negative bands around 290, 375 and 430 nm. The bleaching at 430 nm is very sharp with an extinction coefficient of about 28.5 cmqu4. The PV680/P680 absorption difference spectrum we obtained is very similar to the spectrum recently reported by Gerken and coworkers [1] in cyanobacterium except that they observed two peaks at 400 nm and 415 nm, whereas only one peak at 400 nm is resolved in our spectrum. The spectrum is different from that in trypsinized and NHZOH treated PSII obtained by Weiss and Renger [18], which might be caused by the different method P+ 0n th 122 employed to subtract QA'/QA contributions. Compared to the in vitro spectrum of Chla’/Chla (Figure 4-3, dashed line), there is a red shift of the 400 nm peak of the P”680/P680 spectrum in tris-washed PSII, the bleaching at 430 nm is much sharper, the extinction coefficient at 430 nm is smaller [19,32]. These differences can be explained in terms of the aggregation of the reaction center chlorophyll (presumably a dimer [33]) that lowers the excited state energy and results in a red shift of the spectrum. In addition, the presence of charged species near the reaction center P680 might also play an important role in the modification of the spectrum. Although Brudvig and coworkers [10,43] have suggested the possibility that a nearby chlorophyll, rather than.PV680, is detected in various. of the systems designed to monitor Q{]F”680 recombination, the results we obtained here strongly suggest that the reaction is indeed the charge recombination reaction from the state Q{/P”680. This conclusion is supported by the decay kinetics of QA' and P’680, which show nearly identical decay halftimes. Moreover, the difference spectrum we obtained here is very similar to the P”680/P680 difference spectrum recently reported by Gerken and coworkers [1], which indicates that the reaction center chlorophyll 1’680, rather than the auxiliary chlorophyll, is observed in our experiments. Finally, the similar kinetic behavior and the narrower EPR linewidth of the transient species observed 123 by Hoganson and Babcock [44] by using time resolved EPR technique is also consistent with this assumption. The absorption difference spectrum of P’680/P680 in NHZOH treated tris-PSII shows near identically features to that in tris-washed PSII (Figure 4-4). The detection of the photo- induced charge separated state QA'/P*68O at 325 nm and 820 nm (Figure 3-6 and 3-7 in the chapter III) indicates that the reaction center P680 is still fully functional in NI-IZOH treated samples. The NHZOH treatment does not change the difference spectrum of P’680/P680. This suggests that NHZOH is not involved in direct interaction with the reaction center chlorophylls P680, since modification to the micro-environment should affect the difference spectrum of P‘680/P680. Our results show that the NI-IZOH inhibition site is more likely located near Y2. The reasoning that underlies this conclusion is the following: (1) NHZOH has no effect on the absorption difference spectrum of reaction center P680, nor on the formation of the charge separated state QA'/P*680. (2) the difference spectrum in the millisecond time scale, which arises from the charge recombination reaction between QA' and 22*, is modified by the NHZOH treatment. (3) EPR data [23] show that Y; can not be generated in NHZOH treated samples. The spectrum in Figure 4—5 shows the 45 ms decay phase (open circle) in tris-washed PSII, which arises from the charge recombination between QA' and Y; [3] . Upon NHZOH .u... .___.._.. .ILAI ... - \ In in (101 124 treatment, the amplitude of the spectrum is reduced, and the shape of the spectrum is altered as well. This implies that the electron transfer from Y2 to P’680 is inhibited and that the electron transfer pathway is modified in NHZOH treated tris-PSII. Figure 4-6 shows the spectrum (open circle) after subtraction of the Qflf/QA contribution from the spectrum in Figure 4-5 (open circle) in tris-washed PSII. The spectrum has positive bands around 260, 300, 400, and 440 nm, and a negative band at 430 run. It is similar to the Y{]Yi absorption difference spectrum obtained by Dekker and coworkers [3] and has features similar to that of tyrosine radical in water in the UV region (Figure 4-6). This supports the conclusion that the 45 ms decay phase arises from charge recombination between QA' and Yz*. However, in NHZOH treated tris-PSII, the spectrum after subtraction of the QA'/QA contribution (Figure 4-7) is different from the Yz"/Yz spectrum, especially in the visible region. The components that contribute to this spectrum are not known yet, although it seems that some contributions may arise from the oxidized reaction center EVGBO. other electron donors/acceptors may participate in the electron transfer reactions in NHgni inhibited tris-PSII, but the nature of these electron donors/acceptors were not identified in our experiments. The possibility that NH20H inhibition takes place near the Iz site rather than at the reaction center P680 raises an '1‘. t“ A 125 interesting question as to how NHZOH reacts with Y2 and prevents it from being oxidized. The chemistry of tyrosine in water [34,35] shows that the pK value for H3 release from Tyr-OI-I is 10.1 and the value decreases to -1.6 upon the oxidation of the tyrosine (Tyr-OHf). Thus it is likely that deprotonation of Y1 occurs with electron extraction by'PVGBO in PSII. The EPR data support this supposition as the EPR signal of Yz“ is characteristic of a neutral rather than a cation radical [8]. We expect, then, that protonation/ deprotonation reaction of the tyrosine phenol oxygen will occur during the oxidation and reduction of Y2 during the electron transfer reaction in PSII. Optical evidence to support this model has been observed in inactivated PSII [36,37]. Figure 4-8 shows the scheme of the hydrogen shift during the oxidation and reduction reactions. The results of Eckert and Renger [38] suggest that the hydrogen bond shift, rather than proton release and re-uptake, occurs within a double well potential in intact PSII membrane preparations because the observed 10 kJ/mol activation energy of the electron transfer reaction from Yz to P‘680 is smaller than typical average energies of 20-25 kJ/mol for hydrogen bonds in proteins [39]. However in tris-washed PSII, which lacks oxygen evolution capacity, the activation energy is found to be about 45 kJ/mol [40]. This change of the activation energy can play an important role for the 126 ,9 Q; .9 /‘<\' __x\/ x O O O ———> > (EH2 (sz (EH2 R R R Figure 4-8 The electron transfer and hydrogen shift of the electron donor Y2 during the electron transfer reactions in PSII. Here B represents an amino acid residue that forms a hydrogen bond with the tyrosine. retardation of electron transfer from Y2 to P‘680, which is significantly slowed upon tris inhibition [38]. The inhibition of NHZOH may be the results of a chemical reaction between the NHZOH and the phenol group of the tyrosine residue. The phenol is nucleophilic and may react at oxygen or carbon centers that are neutral or positively charged electrophiles [41] . NHZOH undergoes hydrolysis in aqueous solution to yield N’H30H [42]. since the NHZOH inhibition is reversible [23], covalent bond formation is unlikely during a chemical reaction of NHZOH with Y2. One Pi 127 jpossible reaction that fulfills these criteria is presented in Figure 4-9. In the scheme, the hydroxyl proton of the phenol group of Y2 is shifted upon the extraction of an electron by IVGBO. When it is re-reduced in the presence of protonated lflgDH, an ionic bond between the oxygen and the nitrogen is formed instead of the normal protonation reaction. The formation of the ionic bond inhibits the normal hydrogen bond shift and changes the redox potential of Y2 so that the electron transfer from Y2 to P’680 can not proceed. The light requirement of the NHZOH inhibition in PSII can be explained by the light induced hydrogen bond shift of the phenol group of Y2 that results in the reaction with NHZOH to form the O'-N” ionic bond. OH 0 OGN‘T’H3OH _ n ~ -I/ Q; \ O Q \3 (L) N6H30H 7 H+ I R R R Figure 4-9 The possible chemical reactions of NHZOH with the phenol group of the tyrosine residue of Y2 in PSII. ET"— 128 Conclusion The absorption difference spectrum, of different decay phases can be used to identify components participating in photosynthetic reactions as well as the changes of their micro-environments under different treatments. Our results in tris-washed PSII show that the difference spectrum of the 230 as decay phase of the charge separated state QA'/P*680 arises from the contributions of QA'/QA and P‘680/P680; the spectrum of the 45 ms decay phase arises from the contributions of QA'lQA and Yz*/Yz. NHZOH treatment has no effect on the spectrum of the microsecond decay phase, but the spectrum. of the 'millisecond. decay' phase is altered and diminished. We conclude that the 230 us decay phase arises from the charge recombination reaction of the state Q{/P”680 and the 45 ms decay phase comes from the charge recombination reaction between,Q{ and Yf'in tris-washed PSII. Because NHgni treatment has no effect on the E”680/P680 absorption difference spectrum, the inhibition site of NHZOH is more likely to be near the Y} site rather than near the reaction center P680. The inhibition of the electron transfer capability of Y2 under NHZOH treatments is proposed to involve the reactions of NHZOH with the phenol group of the tyrosine residue. This reaction either prevents the hydrogen shift that is required for the electron transfer to take place or 129 changes the redox potential and electron coupling between Y2 and P’680 so that Yz can no longer donate electrons to P’680. 10. 11. References Gerken, S., Dekker, J.P., Schlodder, E. and Witt, H.T. Biochim. Biophys. Acta 1989, 977, 52 Dekker, J.P., Van Gorkom, H.J., Wensink, J. and Ouwehand, L. Biochim. Biophys. Acta 1984, 767, 1 Dekker, J .P. , Van Gorkom, H.J. , Brok, M. and Ouwehand,L. giochim. Biophys. Acta 1984, 764, 301 Van Gorkom, H.J., Pulles, M.P.J. and Wessels, J.S.C. Biochim. Biophys. Acta 1975, 408, 331 Van Gorkom, H.J. Biochim. Biophys. Acta 1974, 347, 439 Lavergne, J. FEBS Lett. 1984, 173, 9 Diner, B. and Volker, M. (1984) in Advances in Photosynthesis Research (Sybesma, C., ed.) Vol. I. page 407, Martinus Nijhoff/Dr. W. Junk Publishers, Dordrecht, The Netherlands Barry, B.A. and Babcock, G.T. Proc. Natl. Acad. Sci. USA 1987, 84, 7099 Debus, R.J., Barry, B.A., Babcock, G.T. and McIntosh,L. Eroc, Natl. Acad. Sci. USA 1988, 85, 427 Metz, J.G., Nixon, P.J., Rogner, M., Brudvig, G.W. and Diner, B.A. giochemigtry 1989, 28, 6960 Vermaas, W.F.J., Rutherford, A.W. and Hansson, 0. Proc. Natl. Acad. Sci. USA 1988, 85, 8477 130 "“t»sr 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 131 Bent, D.V. and Hayon, E. J. Am. Chen. Soc. 1975, 97, 2599 Bensasson, R.V., Land, E.J. and Truscott, T.G. (1983) Flash Phgtolysis and Pulse Badiolysi's page 93, Pergamon, Oxford Gerken, S., Brettel, K., Schlodder, E. and Witt, H.T. EEBS Let . 1988, 237, 69 Ghanotakis, D.F., O'Malley, P.J., Babcock, G.T. and Yocum, C.F. (1983) in Oxygen Evolving Systems of Plant Photosynthesis (Inoue, Y., ed.) page 91, Academic Press Japan Inc., Tokyo Babcock, G.T., Buttner, W.J., Ghanotakis, D.F., O'Malley, P.J., Yerkes, C.T. and Yocum, C.F. (1984) in Pnogeedings of nhe 6th International Congness on Photosynthesis (Sybesma, C., ed.) Nijhoff/Junk, The Hague Brok, M., De Groot, A. and Hoff, A.J. (1984) in Proceedings of the 6§h Ingennational Congress on Photosynthesis (Sybesma, C., ed.) Nijhoff/Junk, The Hague Weiss, W. and Renger, G. Biochim. Biophys. Acta 1986, 850, 173 Borg, D.C., Fajer, J., Felton, R.H. and Dolphin, D. Proc. Natl. Acad. Sci. USA 1970, 67, 813 Berthold, D.A., Babcock, G.T. and.Yocum, C.F. FEBS Lett. 1981, 134, 231 Ghanotakis, D.F., Babcock, G.T. and‘Yocum, C.F. Biochim. Biophys. Acta 1984, 765, 388 Ford, R.C. and Evans, M.C.W. Biochim. Biophys. Acha 1985, 807, 1 Ghanotakis,D.F. and Babcock,G.T. FEBS Leth. 1983,153,231 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 132 Stiehl, H.H and Witt, H.T. z. Nanunforsch 1968, 23b, 220 Stiehl, H.H and Witt, H.T. Z. Naturforsch 1968, 24b,1588 Wasielewski, M.R., Johnson, D.G., Seibert, M. and Govindjee, Proc. Natl. Acad. Sci. USA, Vol. 86, 524-528, January 1989 Eckert, H.-J, Wiese, N., Bernarding, J., Eichler, H.-J. and Renger, G. FEBS Lett. 1988, 240, 153 Ghanotakis, D.F. and Babcock, G.T. FEB§ILet§. 1983, 153, 231 Ford, R.C. and Evans, M.C.W. FEBS Lett. 1983, 160, 159 Vermeglio, A. and Mathis, P. Biochim. Biophys. Acta 1973, 314, 57 Hoganason, C.W., Babcock, G.T. and Yocum, C.F. Photosyn. Res. 1989, 22, 285 Davis, M.S., Forman, A. and Fajer, J. Proc. Natl. Acad. Sci. USA, 1979, 76, 4170 R. Van Grondelle (1985) , Biochim. Biophys. Acta, 811,147 Dixon, W.T. and.Murphy, D. J. Chem. Soc. Faraday Trans.2 1976, 72, 1221 Barker, R. Organic Chemistry of Biological Compounds (1971) Prentice-Hall, Inc., Englewood Cliffs, New Jersey Renger, G. and V61ker, M. FEBS Lett. 1982, 149, 203 Farster, V. and Junge, W. (1984) in Advances in Photosynthesis Research (Sybesma, C. ed.) Vol. II, 305, Nijhoff/Junk, The Hague Eckert, H.J. and Renger, G. FEBS Let . 1988, 236, 425 $‘————‘ l‘-__ 39. 40. 41. 42. 43. 44. 133 Stearn, A.E. (1949) Adv. Enzynol. 9, 25 Reinman, S. and Mathis, P. Biochim. Biophys. Acta 1981, 635, 249 Whiting, D.A. in Comprehensive Organic Chemistry (Barton,S.D. et. al. ed.) 1979, Vol 1, page 714 Jones, K. in Comprehensiye Inorganic Chemistry (Bailar, J.C. ed.) 1973, Vol 2, page 265 Thompson, L.K. and Brudvig, G.W. Biochemistry 1988, 27, 6653 Hoganson, C.W. and Babcock, G.T. Biochemistny 1989, 28, 1448-1454 CHAPTER V SUMMARY AND FUTURE WORK EBEEA£I The charge separated state QA'lP’680 is created within 300 ps following the excitation of the reaction center chlorophyll P680 by light in photosystem II in higher plants [1,2]. In intact PSII membrane preparations or chloroplasts, this charge separated state decays on the nanosecond time scale by a rapid electron donation by the physiological donor Yz [3w41- Certain chemical treatments that inhibit oxygen evolution or electron transfer reactions on the donor side of PSII stabilize P’680 so that the lifetime ‘of the state QA'/P*680 is extended into the microsecond time range. The time evolution of Q{/P”680 is highly dependent on the micro- environments of the electron carriers and the presence of inhibitors. Studies of the decay kinetics of Q{]I”680 can provide us with valuable information about the mechanism of the inhibition reactions as well as the modifications of the micro-environments under different treatments. In the experiments described in this dissertation, we investigated the kinetic and spectroscopic properties of the 134 135 reaction center P680, primary donor Y2, and acceptor QA in tris-washed, NaCl washed, and NHZOH treated PSII by monitoring the decay of the charge separated state Qg/PV680 with transient absorption spectroscopy. The results we obtained can be summarized as follows: (1) The charge recombination reaction between QA' and P‘680 occurs in the microsecond time range in tris-washed and NaCl-washed PSII under high flash repetition rate. NHZOH treatment removes the high flash repetition rate requirement for observing Q{/P”680 recombination. ( 2) The rate of the charge recombination reaction between Q" and P‘680 in NaCl washed PSII (halftime z 130 us) is about twice as fast as that in tris-washed PSII (halftime:z 230 ns). (3) The secondary plastoquinone acceptor QB is not directly involved during the charge recombination reactions. The QA to QB electron transfer reaction, which presumably has a halftime of 200 us, was not observed. (4) In tris-washed PSII, the absorption difference spectrum of the 230 us decay phase of the charge separated state QA'/P”680 arises from the contributions from QA‘/QA and P‘680/P680; the spectrum of the 45 ms decay phase arises from contributions from QA'/QM and Yz*/Yz. (5) NHZOH treatment has no effect on the absorption difference spectrum of the microsecond decay phase, but the absorption difference spectrum of the millisecond decay phase is altered and diminished. Based on the above results, as well as on observations CE re 136 from other laboratories [4-11], we conclude the following: (1) The mode of tris inhibition is distinct from that of NHZOH treatment. Tris washing inhibits the re-reduction of Yz" by its physiological donor, whereas the electron transfer from Y2 to P‘680 is not affected greatly. NHZOH treatment greatly retards the electron transfer from Y2 to P‘680 and keeps Y2 in its reduced state. (2) The difference in the decay rate of the charge recombination reaction between QA' and P‘680 in NaCl washed PSII and in tris-PSII probably arise from different micro-environments of reaction center P680 in the two preparations. (3) The absence of direct involvement of the secondary plastoquinone acceptor QB in oxidizing q" in the QA' /P’680 state may be attributed to the effects of the positive charge on the reaction center P‘680 or to the removal of Q8 molecules during sample preparation. ( 4) In tris-washed PSII, the 230 us decay phase arises from charge recombination between QA' and P‘680, and the 45 ms decay phase arises from charge recombination involving QA' and Yz‘. ( 5) The Soret band absorption red shift in the difference spectrum of P‘680/P680 relative to that of in vitro Chla’/Chla presumably arises from the aggregation state of the reaction center chlorophylls. (6) The inhibitory site of NHZOH is more likely located near Y2 than near P680. The inhibition of the electron transfer capability of Y2 under NHZOH treatments may involve reversible reaction of NHZOH with the phenol group of the tyrosine 137 residue to form an Ofidr ionic bond. In such scenario this reaction would disrupt the network of covalent and hydrogen bonds and change the redox properties of Y: so that electron transfer from Y2 to P‘680 can no longer be carried out effectively in competition with recombination. gutnge Work Transient absorption spectroscopy has been a very useful technique for studying electron transfer reactions that occur in photosynthesis. Almost all electron transfer components of photosystem II exhibit absorption changes upon oxidation or reduction following flash excitation. Thus, they are detectable by transient absorption spectroscopy. The studies presented in this dissertation show that.we are able to obtain kinetic and spectroscopic information of individual electron transfer components in PSII by using the transient absorption technique. Spectral overlap of different components, which causes difficulties in interpreting' the results, can. be reduced by carefully preparing the reaction assay so that the oxidation or reduction of different components take place on different time scales. Our results show that the charge recombination reaction between QA' and P’680 occurs when the physiological electron transfer reactions is inhibited by tris, NaCl, or NHZOH treatment. However, there have been suggestions that other mC 138 alternative donors, besides the Y2, may exist and donate electrons to EV680 when the physiological electron transfer reactions are inhibited. There have been many reports that non-physiological electron donors, such as chlorophylls [12-15], cytochrome b559 [16,17], and carotenoid [18], can be oxidized by P”680 under certain conditions. It is worthwhile to investigate further these reactions by using transient absorption technique. By monitoring the charge recombination and absorption difference spectra in PSII 'membrane preparations under different inhibitory treatments, we should achieve a better understanding as to how these alternate donors participate in the electron transfer reactions under certain conditions. The micro-environment plays a very important role in the electron transfer reactions in PSII. This has been observed in our experiments and in several other laboratories [5,6]. The QA' to Q3 electron transfer is greatly modified in tris- washed.PSII5 The charge recombination reaction between.Q{’and I”680 in NaCl washed PSII is faster than that in tris-washed PSII because of the effects of the micro-environment. However, no extensive studies have been performed in such a way that allow'us to obtain detailed information about hOW'the electron transfer reactions are affected by the changes of the micro-environment” Mutagenesis techniques, which have become more and more important in photosynthesis research, may be 139 employed to address these reactions in greater detail. By selectively replacing certain redox active amino acid residues in PSII membrane polypeptides, and by monitoring the reaction rate and by measuring the induced free energy changes, we should be able to establish correlations between the function of individual components and the rate of electron transfer. Protonation and deprotonation of the phenol head group have been suggested to be essential in the electron transfer reactions of Y2 in PSII [19,20]. The results presented in chapter IV suggest that NHZOH inhibition may be the result of the inhibition of the protonation/deprotonation reaction of Y2. Since protonation/deprotonation is directly affected by the pH value of the solution, the charge recombination in tris-washed PSII should be strongly depend on pH, and this pH dependence may be modified in NHZOH treated PSII. Thus, studying the pH dependence of the charge recombination in tris-washed PSII and NHZOH treated tris-PSII may provide useful information on the importance of protonation/ deprotonation reactions that may occur during electron transfer and the mode of NHZOH inhibition in blocking this proton motion. 10. 11. 12. 13. 14. Referenceg M.R. Wasielewski, D.G. Johnson, M. Seibert, and Govindjee, Proc. Natl. Acad. Sci. USA, Vol. 86, 524-528, January 1989 Eckert, H.-J, Wiese, N., Bernarding, J., Eichler, H.-J. and Renger, G. FEBS Le; . 1988, 240, 153 Brettel,K. , Schlodder,E. , and Witt,H.T. Biochim. Biophys. Acta 766, 1984, 403 Gerken,S., Brettel,K., Schlodder,E., and Witt,H.T. FEBS Lett. 1988, 237, 69 Gerken, S., Dekker, J.P., Schlodder, E. and Witt, H.T. Biochim. Biophys. Acta 1989, 977, 52 Ford, R.C. and Evans, M.C.W. Biochim. Biophys. Acta 1985, 807, 1 Hoganson, C.W. and Babcock, G.T. Biochemistry 1988, 27, 5848 Ghanotakis, D.F. and Babcock, G.T. FEBS Lett. 1983, 153, 231 Dekker, J.P., Van Gorkom,H.J., Brok, M. and Ouwehand,L. Biochim. Biophys. Acta 1984, 764, 301 Weiss, W. and Renger, G. Biochim. Biophys. Acta 1986, 850, 173 Eckert, H.J. and Renger, G. FEBS Legh. 1988, 236, 425 Visser,J.W.M., Rijersberg,C.P. and Gast,P. B'o ' . Elgphys. Acta 460, 1977, 36 De Paula,J.C., Innes,J.B. and Brudvig,G.W. Bigghgninhny VOl. 24, 1985, 8114 Thompson, L.K. and Brudvig, G.W. Biochemistry 1988, 27, 6653 140 .n‘. 15. 16. 17. 18. 19. 20. Metz, J.G., Nixon, P.J., Rogner, M, Brudvig, G.W. and Diner, B.A. Biochemistry 1989, 28, 6960 Vermeglio,A. and Mathis,P. Biochim. Biophys. Acta 314, 1973, 57 Malkin,R. and vanngard,T. FEBS Lett. 111, 1980, 228, 16. Mathis,P. and Rutherford,A.W. Biochim. Biophys. Acta 767, 1984, 217 Mathis, P. and Rutherford, A.W. Biochin. Bignhys. Acta 767, 1984, 217 Renger, G. and volker, M. FEBS Lett. 1982, 149, 203 Fdrster, V. and Junge, W. (1984) in Advances in Phgtosynthesis Research (Sybesma, C. ed.) Vol. II, 305, Nijhoff/Junk, The Hague 141 c ) " 5‘-.._.__._ -. n. ' I ‘ APPENDICES APPENDIX A SOURCE CODES OF PROGRAM TASCI '*********************************************************** PROGRAM TASCI By Xingmin Liu Dept. of Chemistry Michigan State University April 5, 1988 PROGRAM TASCI (TRANSIENT ABSORPTION SPECTROMETER COMPUTER INTERFACE) CONTROLS THE DATA ACQUISITION,TIMING SEQUENCES AND INSTRUMENT INTERFACE. IT WAS DESIGNED TO OPERATE THE TRANSIENT ABSORPTION SPECTROMETER BUILT BY D. LILLIE AND XINGMIN LIU FOR STUDIES OF THE ELECTRON TRANSFER REACTIONS IN PHOTOSYNTHESIS. ’ I '*********************************************************** 100 I---- PARAMETER INPUT ----- 110 A% = 16000 120 DIM DAT!(A%) 140 CLS 200 WAS = "WAVELENGTH (nm) " 210 PRINT WA$; : INPUT WAVL% 220 SR$ = "SAMPLING RATE (us) " 230 PRINT SR$; : INPUT SR! 240 IF SR1 < .05 OR SR! > 80000.1 THEN PRINT " ****** BAD SAMPLING RATE ******" GOTO 230 ELSE GOTO 250 END IF 250 NS$ = "NUMBER OF SAMPLES " 260 PRINT NS$; : INPUT ; NS% 142 270 280 290 300 310 315 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 600 610 620 630 640 700 710 143 IF (.001 * SR! * NS%) > 8000.1 0R NS% > 16000 THEN PRINT " T00 MANY SAMPLES" GOTO 230 ELSE GOTO 280 END IF PRINT USING " t = ####.### ms"; .001 * SR * NS% NA$ = "NUMBER OF AVERAGES " PRINT NA$; : INPUT NA% IF NA% = 0 THEN NA% = 1 NA1% = NA% INPUT "NUMBER OF SCANS TO BE DISPLAYED ON SCREEN "; SCR% IF SCR% = 0 THEN SCR% = 1 DC$ = "DC OFFSET (v) " PRINT DC$; : INPUT DC! CT1$ = "TRIGGERING THE LASER (ms) " GAN$ = "GAIN OF THE PRE-ADC AMPLIFIER " PMT$ = "THE PMT VOLTAGE (v) " CT4$ = "TIME DELAY OF COUNTER 4 (ms) " PRINT GAN$; : INPUT GAN% PRINT PMTS; : INPUT PMT% PRINT CT1$; : INPUT CTl! CT1P! = CTl! CT1& = 1000 * CT1! CT2& = SR! * CLNG(NS%) + 100 'SAMPLING TRIGGER DELAY CT3& = CT1& / 4 'HOLDING TRIGGER DELAY PRINT CT4$; : INPUT CT4! CT4P! = CT4! CT4& = 1000*CT4! I . ------- INPUT END ------------ GOTO 1000 'SET TIMER AND ADC BOARD, START MEASUREMENT n . -------- COMMAND INPUT ----------- I LOCATE 25, 1: COLOR 12 PRINT "COMMAND: Save Reset Do Continue Agan Plot View Window Exit"; COLOR 15, 9: LOCATE 1, 1 KEY$ = INKEY$ SELECT CASE KEY$ CASE "S", "S" GOTO 3000 CASE "R", urn FOR I = 0 TO NS% DAT!(I) = 0 NEXT I GOTO 200 CASE "D", udu 144 FOR I = 0 TO NS% DAT!(I) = 0 NEXT I L% = 0 GOTO 2030 CASE "C", "C" GOTO 2030 CASE "A", "a" NA% = NA% + NA1% GOTO 2030 CASE up", up" GOTO 6000 CASE "V", "VI: GOTO 4000 CASE "W", uwu GOTO 5000 CASE "E", "en SCREEN 0: STOP CASE ELSE 750 END SELECT 760 GOTO 700 800 END 900 ' 1000 '***************** SET THE TIMER ******************** 1010 ' 1020 ' ---------- SET THE MASTER MODE --------- 1030 OUT 769, 23 1040 OUT 768, 0 1050 OUT 768, 145 1060 ' 1100 ' ------ SET THE SOFTWARE TRIGGER --------- 1110 OUT 769, 1 1120 OUT 768, 5 1130 OUT 768, 11 1140 OUT 769, 9 1150 OUT 768, 250 'A/D TRIGGER DELAY FOR DCR-lA 1160 OUT 768, 0 1170 ' 1200 ' ------ SET THE TIMNE DELAY COUNTERS -------- 1210 SELECT CASE CT1& 'SELECT THE CLOCK SPEED CASE 0 TO 50000 M1% = 11 CT1& = CT1& CASE 50001 TO 500000 M1% = 12 CT1& = .1 * CT1& CASE 500001 TO 5000000 M1% = 13 CT1& = .01 * CT1& CASE 5000001 TO 50000000 1250 1260 1270 1280 1300 1350 1360 1370 1380 1400 145 M1% = 14 CT1& = .001 * CT1& CASE 50000001 TO 60000000 M1% = 15 CT1& = .0001 * CT1& CASE ELSE PRINT "THE LASER TRIGGER OUT OF THE RANGE" GOTO 420 END SELECT OUT 769, 2 OUT 768, 1 OUT 768, M1% SELECT CASE CT2& CASE 0 TO 10000 M2% = 11 CT2& = CT2& CASE 10001 TO 100000 M2% = 12 CT2& = .1 * CT2& CASE 100001 TO 1000000 M2% = 13 CT2& = .01 * CT2& CASE 1000001 TO 10000000 M2% = 14 CT2& = .001 * CT2& CASE 10000001 TO 60000000 M2% = 15 CT2& = .0001 * CT2& CASE ELSE PRINT "THE SAMPLING TRIGGER OUT OF THE RANGE " GOTO 420 SELECT 769, 3 OUT 768, 5 OUT 768, M2% SELECT CASE CT3& CASE 0 TO 10000 M3% = 11 CT3& = CT3& CASE 10001 TO 100000 M3% = 12 CT3& = .1 * CT3& CASE 100001 TO 1000000 M3% = 13 CT3& = .01 * CT3& CASE 1000001 TO 10000000 M3% = 14 CT3& = .001 * CT3& CASE 10000001 TO 60000000 M3% = 15 END OUT 1450 1460 1470 1480 1500 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650 1700 1710 1720 1730 1740 1800 1810 1820 1830 1840 1900 CT3& 146 .0001 * CT3& CASE ELSE END OUT OUT OUT PRINT "THE HOLDING TRIGGER OUT OF THE RANGE" GOTO 420 SELECT 769, 768, 768, 4 5 M3% SELECT CASE CT4& CASE 0 TO 10000 M48 = 11 CT4& = CT4& CASE 10001 TO 100000 M48 = 12 CT4& = .1 * CT4& CASE 100001 TO 1000000 M48 = 13 CT4& = .01 * CT4& CASE 1000001 TO 10000000 M48 = 14 CT4& = .001 * CT4& CASE 10000001 TO 60000000 M48 = 15 CT4& = .0001 * CT4& CASE ELSE CLS PRINT IICOUNTER 4 OUT OF THE RANGEII GOTO 470 END SELECT OUT 769, 5 OUT 768, 5 OUT 768, M48 I I ------- LOAD THE TIME DELAY COUNTERS ---------- CTIH8 = INT(CT1& / 256) CT1L8 = INT(CT1& - 256 * CLNG(CT1H8)) OUT 769, 10 OUT 768, CT1L8 OUT 768, CT1H8 CT2H8 = INT(CT2& CT2L8 OUT 769, OUT 768, CT2L8 OUT 768, CT2H8 CT3H% = INT(CT3& CT3L% = INT(CT3& OUT 769, OUT 768, CT3L% OUT 768, CT3H% CT4H% = INT(CT4& / \ 256) INT(CT2& 256 * CLNG(CT2H8)) 11 \ 256) 256 * CLNG(CT3H8)) 12 256) 1910 1920 1930 1940 1950 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100 147 CT4L8 = INT(CT4& - 256 * CLNG(CT4H8)) OUT 769, 13 OUT 768, CT4L8 OUT 768, CT4H8 I I************** DATA ACQUISITION ***************** I L8 = 0 SCREEN 9 COLOR 15, 9 WINDOW (-.05 * NS8, -145)-(1.05 * NS%, 145) M38 = INT(NS8 / 1000) IF MS8 = 0 THEN M38 = 1 LOCATE 1, 1 PRINT NA$; NA8 SELECT CASE SR! CASE .05 CTRL8 = 0 DATMULP8 = 1 CASE .1 CTRL8 = 0 DATMULP8 = 2 CASE .2 CTRL8 = 0 DATMULP8 = 4 CASE .5 CTRL8 = 16 DATMULP8 = 1 CASE 1 CTRL8 = 16 DATMULP8 = 2 CASE 2 CTRL8 = 16 DATMULP8 = 4 CASE 5 CTRL8 = 32 DATMULP8 = 1 CASE 10 CTRL8 = 32 DATMULP8 = 2 CASE 20 CTRL8 = 32 DATMULP8 = 4 CASE 50 CTRL8 = 48 DATMULP8 = 1 CASE 100 CTRL8 = 48 DATMULP8 = 2 CASE 200 'SELECT SAMPLING RATE 148 CTRL% = 48 DATMULP% = 4 CASE 500 CTRL% = 64 DATMULP% = 1 CASE 1000 CTRL% = 64 DATMULP% = 2 CASE 2000 CTRL% = 64 DATMULP% = 4 CASE 5000 CTRL% = 64 DATMULP% = 10 CASE 10000 CTRL% = 64 DATMULP% = 20 CASE 20000 CTRL% = 64 DATMULP% = 40 CASE 50000 CTRL% = 64 DATMULP% = 100 CASE 80000 CTRL% = 64 DATMULP% = 160 CASE ELSE CLS PRINT "NON-AVAILABLE SAMPLING RATE" GOTO 230 2120 END SELECT 2200 NSM% = NS% * DATMULP% 2210 SELECT CASE NSM% 'SELECT NUMBER OF SAMPLES CASE 100 TO 250 NSH% = 254 GOTO 2300 CASE 251 TO 500 NSH% = 253 GOTO 2300 CASE 501 TO 750 NSH% = 252 GOTO 2300 CASE 751 TO 1000 NSH% = 251 GOTO 2300 CASE 1001 TO 1250 NSH% = 250 GOTO 2300 CASE 1251 TO 1500 NSH% = 249 2250 2260 2300 2310 2320 2330 2340 GOTO 2300 CASE 1501 TO 1750 NSH% = 248 GOTO 2300 CASE 1751 TO 2000 NSH% = 247 GOTO 2300 CASE 2001 TO 2500 NSH% = 245 GOTO 2300 CASE 2501 TO 5000 NSH% = 235 GOTO 2300 CASE 5001 TO 7500 NSH% = 225 GOTO 2300 CASE 7501 TO 10000 NSH% = 215 GOTO 2300 CASE 10001 TO 12500 NSH% = 205 GOTO 2300 CASE 12501 TO 15000 NSH% = 195 GOTO 2700 CASE 15001 TO 16000 NSH% = 192 GOTO 2700 CASE ELSE CLS 149 PRINT"SAMPLING RATE OR NNMBER OF SAMPLES TOO LARGE" GOTO 230 END SELECT I L8 = L8 + 1 DEF SEG = &HD000 POKE &HC002, 99 POKE &HCOOO, 0 POKE &HC001, NSH8 POKE &HCOOO, 0 POKE &HC001, NSH8 POKE &HC002, CTRL% OUT 769, 125 BEGINING SAMPLING 'FOR 100 TO 12500 SAMPLES ’ 'BACKGROUND SCAN 'LOAD THE COUNTER IF PEEK(&HC002) AND 1 THEN 2340 ELSE GOTO 2330 POKE &HC002, 99 N8 = 256 * PEEK(&HC001) + PEEK(&HCOOO) FOR I = 0 TO NS% DAT!(I) = DAT!(I) — PEEK(N8) 'TRANSFER THE 'COLLECTED DATA INTO 2400 2410 2420 2510 2600 ---- GRAPHIC DISPLAY (REAL TIME) ---- GRAPHIC DISPLAY (FINAL) 150 'COMPUTER MEMORY 'AS BACKGROUND DATA 'SIGNAL SCAN N8 = N8 + DATMULP8 NEXT I DEF $86 = &HDOOO POKE &HC002, 99 POKE &HCOOO, 0 POKE &HC001, NSH8 POKE &HC000, O POKE &HC001, NSH8 POKE &HC002, CTRL8 OUT 769, 127 IF PEEK(&HC002) AND 1 THEN 2420 ELSE GOTO 2410 POKE &HC002, 99 N8 = 256 * PEEK(&HC001) + PEEK(&HC000) FOR I = 0 TO NS8 ITRANSFER THE DATA DAT!(I) = DAT!(I) + PEEK(N8) ICOLLECTED INTO N8 = N8 + DATMULP8 ICOMPUTER MEMORY 'LOAD THE COUNTER NEXT I 'AS REAL DATA IF INKEY$ "S" THEN 2600 'CHECK THE INTERRUPT IF INKEYS "s" THEN 2600 IF INT(L% / SCR%) = L% / SCR% OR L% = NA% THEN 2500 ELSE 2300 CLS LOCATE 5, 58: PRINT "CYCLES ="; L%; LOCATE 14, 65: PRINT "t ="; PRINT USING "####.###";.001 * SR * NS8;:PRINT "ms"; FOR I = 0 TO NS8 STEP MS8 Y = DAT!(I) / L8 PSET (I, Y) NEXT I IF L8 < NA8 GOTO 2300 LINE (0, 0)-(NS8, 0), 12, , &HCCCC LINE (0, 40)-(NS8, 40), 9, , &HCCCC LINE (0, 80)-(NS8, 80), 9, , &HCCCC LINE (0, 120)-(NS8, 120), 9, , &HCCCC LINE (0, -40)-(NS8, -40), 9, , &HCCCC LINE (0, -80)-(NS8, -80), 9, , &HCCCC LINE (0, -120)-(NS8, -120), 9, , &HCCCC GOTO 600 CLS LOCATE 5, 58: PRINT "CYCLES ="; L8 LOCATE 14, 65: PRINT "t ="; PRINT USING "####.###";.001 * SR * NS8;:PRINT "ms"; FOR I = 0 TO NS8 STEP MS8 Y = DAT!(I) / L8 PSET (I, Y) NEXT I 2610 2700 2710 2720 2730 2800 2810 2820 151 LINE (0, LINE (0, LINE (0, LINE (0, LINE (0, LINE (0, LINE (0, GOTO 600 0)-(NS8, 0), 12, , &HCCCC 40)-(NS8, 40), 9, , &HCCCC 80)-(NS8, 80), 9, , &HCCCC 120)-(NS8, 120), 9, , &HCCCC -40)—(NS8, -40), 9,., &HCCCC -80)-(NS8, -80), 9, , &HCCCC -120)-(NS8, -120), 9, , &HCCCC ---- FOR 12501 TO 16000 SAMPLES ------ L8 = L8 + 1 DEF SEG = &H0000 POKE &HC002, 99 POKE &HC000, O POKE &HC001, NSH8 POKE &HC000, 0 POKE &HC001, NSH8 POKE &HC002, CTRL8 OUT 769, 125 IF PEEK(&HC002) AND 1 THEN 2630 ELSE GOTO 2620 POKE &HC002, 99 N8 = 256 * PEEK(&HC001) + PEEK(&HCOOO) FOR I = 0 TO NS8 DAT!(I) = DAT!(I) N8 = N8 + DATMULP8 IF N8 > 16383 THEN N8 = 0 NEXT I DEF SEG = &HDOOO POKE &HC002, 99 POKE &HC000, 0 POKE &HC001, NSH8 POKE &HC000, 0 POKE &HC001, NSH8 POKE &HC002, CTRL8 OUT 769, 127 IF PEEK(&HC002) AND 1 THEN 2820 ELSE GOTO 2810 POKE &H0002, 99 N8 = 256 * PEEK(&HC001) + PEEK(&HCOOO) FOR I = 0 TO NS8 DAT!(I) = DAT!(I) + PEEK(N8) N8 = N8 + DATMULP8 IF N8 > 16383 THEN N8 = 0 'BACKGROUND SCAN 'LOAD THE COUNTER - PEEK(N8) 'SIGNAL SCAN 'LOAD THE COUNTER NEXT I I ------ GRAPHIC DISPLAY ---------- CLS LOCATE 5, 58: PRINT "CYCLES ="; L8 LOCATE 14, 65: PRINT "t ="; PRINT USING "####.###";.001 FOR I = 0 TO NS8 STEP MS8 * SR * NS%;:PRINT "ms"; 2910 3000 3010 3020 3030 3040 3050 3060 3070 3080 3090 3100 3110 3120 3130 3140 3150 3160 3170 3180 3190 3200 3210 3220 3230 3240 3250 3260 3270 3280 3290 3300 3310 3320 3330 3340 3350 3360 152 Y = DAT!(I) / L8 PSET (I, Y) NEXT I IF L8 < NA8 GOTO 2700 LINE (0, 0)-(NS8, 0), 12, , &HCCCC LINE (0, 40)-(NS8, 40), 9, , &HCCCC LINE (0, 80)-(NS8, 80), 9, , &HCCCC LINE (0, 120)-(NS8, 120), 9, , &HCCCC LINE (0, -40)-(NS8, -40), 9, , &HCCCC LINE (0, —80)-(NS8, -80), 9, , &HCCCC LINE (0, -120)-(NS8, -120), 9, , &HCCCC GOTO 600 I************* SAVE THE DATA ******************** I INPUT N5 = NS + OPEN "0", #1, N$ PRINT #1, II*****************************************" PRINT #1, " " PRINT #1, DATES, TIMES PRINT #1, " " PRINT #1, "EXPERIMENTAL PARAMETERS" PRINT #1, " " PRINT #1, WA$; WAVL8 PRINT #1, SR$; SR! PRINT #1, NSS; NS8 PRINT #1, NAS; L8 PRINT #1, DC$; DC! PRINT #1, GAN$; GAN8 PRINT #1, PMT$; PMT8 PRINT #1, "(SOFTWARE TRIGGER DELAY PRINT #1, CT1$; CTlP! PRINT #1, CT4$; CT4P! PRINT #1, " " PRINT #1, "*******************II:*********************" PRINT #1, " " PRINT #1, "DATA" PRINT #1, " " PRINT #1, WAVL8 PRINT #1, SR! PRINT #1, N88 PRINT #1, L8 PRINT #1, DC! PRINT #1, GAN8 PRINT #1, PMT8 PRINT #1, CT1P! PRINT #1, CT4P! PRINT #1, "DATA BEGINS HERE" I "ENTER DATA FILE NAME ".DAT" II, N$ 'SAVE THE PARAMETERS 250 us)" 3400 3410 3420 3500 3510 3520 3600 4000 4010 4020 4030 4040 4050 4060 4070 4080 4100 4110 4120 4130 4140 4150 4160 4170 4180 4190 4200 4210 4220 4230 4240 4250 4260 4270 4280 4290 4300 4400 4410 4420 4430 4440 4450 4460 4470 4500 4510 4520 4530 4540 153 FOR I = 0 TO NS8 ISAVE THE DATA PRINT #1, .005 * (DAT1(I) / L8) NEXT I CLOSE #1 PRINT "DATA HAVE BEEN SAVED IN FILE "; N$ GOTO 600 '****************** VIEW DATA ********************** I LOCATE 1, 1 INPUT "SELECT VIEW WINDOW [t1,t2] ", X11, x21 WN1 = X21 - X11 SCREEN 9 COLOR 15, 9 WINDOW ((X11 - .05 * WN), -.74)-((X21 + .05 * WN), .8) CLS T0 = X1: TM = x2 LINE (X11, 0)-(X21, 0), 12, , &HCCCC LINE (X21, 0)-(x21, .04), 12 LINE ((x11 + WN / 2), 0)-((X11 + WN / 2), .04), 12 LOCATE 14, 70: PRINT USING "####.### ms"; TM; LOCATE 14, 5: PRINT USING "t =###.### ms"; X1; LOCATE 14, 36: PRINT USING "####.### ms"; X1 + WN / 2; LOCATE 10, 4: PRINT " + 0.2 v"; LOCATE 7, 4: PRINT " + 0.4 v"; LOCATE 4, 4: PRINT " + 0 .6 , LOCATE 17, 4: PRINT " — 0.2 v"; LOCATE 20, : PRINT " - 0.4 v"- LOCATE 23, : PRINT " - 0.6 v"; LINE (x11, -.64)-(X11, .64) LINE ((X11 + .12 * WN), .2)—(x21, .2), 9, , &HCCCC LINE ((x11 + .12 * WN), .4)-(x21, .4), 9, , &HCCCC LINE ((X11 + .12 * WN), -.2)-(X21, -.2), 9, , &HCCCC LINE ((x11 + .12 * WN), -.4)-(x21, -.4), 9, , &HCCCC LINE ((X11 + .12 * WN), .6)—(X21, .6), 9, , &HCCCC LINE ((X11 + .12 * WN), -.6)-(X21, -.6), 9, , &HCCCC I B8 = 1000 * X11 / SR1: E8 = 1000 * X21 / SR1 NSL8 = 1000 * WN1 / SR1: XINC1 = WN! / NSL8: XL1 = X11 FOR I = 88 TO E8 Y1 = .005 * (DAT1(I) / L8) PSET (XL1, Y1) XL1 = XL1 + XINC1 NEXT I I LOCATE 4, 50 PRINT "WAVELENGTH (nm) "; WAVL8 LOCATE 5, 50 PRINT "SAMPLING RATE (us) "; SR1 LOCATE 6, 50 4550 4560 4570 4600 4700 5000 5010 5020 5030 5040 5050 5060 5070 5080 5100 5120 5130 5140 5150 5160 5170 5180 5200 5210 5220 5230 5240 5250 5260 5270 5280 5290 5300 5310 5320 5330 5400 154 PRINT "NUMBER OF AVERAGES "; L8 LOCATE 7, 50 PRINT "DC OFFSET (v) "; DC1 GOTO 600 I I************** WINDOW ******************** I LOCATE 1, 1 INPUT "SELECT WINDOW [t1,t2,V(min),V(max)] ", X11, x21, Y11, Y21 WN1 = x21 - X11: YM1 = Y21 - Y11 SCREEN 9 COLOR 15, 9 WINDOW ((X11 - .05 * WN), Y11 - .05 * YM1)-((x21 + .05 * WN), Y21 + .05 * YM1) CLS T0 = X11: TM = X21 LINE (X21, O)-(X21, .03 * YM1), 12 LINE ((X11 + WN / 2), 0)-((X11 + WN / 2),.03 * YM1), 12 IF Y11 < 0 AND Y21 > 0 THEN CSL8 = 1 + Y21 * 25 / YM1 LINE (X11, 0)—(X21, 0), 12, , &HCCCC LINE (X21, 0)-(X21, .03 * YM1), 12 LINE ((X11 + WN/2),0)-((x11 + WN / 2),.03 * YM1),12 ELSE CSL8 = 23 YL1 = Y11 + .01 * YM1 LINE (X11, YL1)-(X21, YL1), 12, , &HCCCC LINE (X21, YL1)-(X21, YL1 + .05 * YM1), 12 LINE ((X11 + WN / 2), YL1)-((x11 + WN / 2), YL1 + .05 * YM1), 12 END IF LOCATE CSL8, 70: PRINT USING "####.### ms"; TM; LOCATE CSL8, 1: PRINT USING "####.### ms"; x1; LOCATE CSL8, 36: PRINT USING "####.### ms"; x1 + WN /2; LINE (X11, Y11)-(x11, Y21) LINE (X11, .1)-(X21, .1), 9, , &HCCCC LINE (X11, .2)-(X21, .2), 9, , &HCCCC LINE (x11, .3)-(x21, .3), 9, , &HCCCC LINE (X11, .4)-(X21, .4), 9, , &HCCCC LINE (X11, .5)-(X21, .5), 9, , &HCCCC LINE (X11, .6)—(X21, .6), 9, , &HCCCC LINE (X11, -.1)-(x21, -.1), 9, , &HCCCC LINE (X11, -.2)-(X21, -.2), 9, , &HCCCC LINE (x11, -.3)-(X21, -.3), 9, , &HCCCC LINE (X11, -.4)-(X21, -.4), 9, , &HCCCC LINE (X11, -.5)-(X21, -.5), 9, , &HCCCC LINE (X11, -.6)-(X21, -.6), 9, , &HCCCC I 88 = 1000 * X11 / SR1: E8 = 1000 * X21 / SR1 5410 5420 5430 5440 5450 5460 5470 5500 5510 5520 5530 5540 5600 5700 6000 6010 6020 6030 6040 6050 6100 6110 6120 6130 6140 6150 6160 6170 6180 6190 6200 6210 6220 6230 6240 6250 6260 6270 6280 6290 6300 6310 6320 6330 6340 6350 6400 6410 6420 6430 155 NSL8 = 1000 8 WN1 / SR1: XINC1 = WN1 / NSL8: XL1 = X11 FOR I = 88 TO E8 Y1 = .005 8 (DAT1(I) / L8) PSET (XL1, Y1) XL1 = XL1 + XINC1 NEXT I I LOCATE 3, 5 PRINT USING " +##.### v"; Y21; LOCATE 23, 5 PRINT USING " +##.### v"; Y11; I GOTO 600 I '****************** I SCREEN 9 COLOR 15, 9 WINDOW (—.05 8 NS8, CLS LINE (0, LINE (NS8, LINE (NS8 / 2, PLOT DATA ********************** -.74)-(1.05 8 NS8, .8) 0)-(NS8, 0), &HCCCC O)-(NS8, .04), 0)-(NS8 / 12. . 12 2. .04), 12 LOCATE 14,70:PRINT USING "###.### ms";.001 8 SR1 8 NS8; LOCATE 13, 8: PRINT "0.0 v"; LOCATE 14,36:PRINT USING "###.### ms";.0005 8 SR1 8 NS8 LOCATE 10, 4: PRINT " + 0.2 v"; LOCATE 7, 4: PRINT " + 0.4 v"; LOCATE 4, 4: PRINT " + 0.6 v"; LOCATE 17, 4: PRINT " - 0.2 v"; LOCATE 20, 4: PRINT " - 0.4 v"- LOCATE 23, 4: PRINT " - 0.6 v"; LINE (0, -.64)-(0, .64) LINE (.12 8 NS8, .2)-(NS8, .2), 9, , &HCCCC LINE (.12 8 NS8, .4)-(NS8, .4), 9, , &HCCCC LINE (.12 8 NS8, -.2)-(NS8, -.2), 9, , &HCCCC LINE (.12 8 NS8, -.4)-(NS8, —.4), 9, , &HCCCC LINE (.12 8 NS8, .6)-(NS8, .6), 9, , &HCCCC LINE ( 12 8 NS8, -.6)-(NS8, -.6), 9, , &HCCCC I IF NS8 > 640 THEN MP8 = INT(NS8 / 640) ELSE MP8 = 1 FOR I = 0 TO NS8 STEP MP8 Y1 = .005 8 (DAT1(I) / L8) PSET (I, Y1) NEXT I I LOCATE 4, 50 PRINT "WAVELENGTH (nm) "; WAVL8 LOCATE 5, 50 PRINT "SAMPLING RATE (us) "; SR1 6440 6450 6460 6470 6480 6500 156 LOCATE 6, 50 PRINT "NUMBER OF AVERAGES "; L8 LOCATE 7, 50 PRINT "DC OFFSET (v) "; DC1 I GOTO 600 '** I '** 100 110 120 130 140 150 160 170 180 190 200 210 220 230 250 260 270 280 290 APPENDIX B SOURCE CODES OF PROGRAM DTDSP ********************************************************* PROGRAM DTDSP By Xingmin Liu Dept. of Chemistry Michigan State University April 10, 1988 PROGRAM DTDSP RETRIEVES THE DATA SAVED BY THE PROGRAM TASCI FROM DISK,TRANSFERS THE DATA INTO ABSORBANCE UNITS, DISPLAYS THE DATA ON THE COMPUTER MONITOR, AND TRANSFERS THE DATA INTO KFIT, PLOTIT, AND PFF FORMATS. ********************************************************* IRETRIEVE THE DATA A8 = 8000 DIM DAT1(A8): DIM BDAT1(A8) CLS LOCATE 4, 1: SHELL "DIR *.DAT/W " LOCATE 1, 1: PRINT " " LOCATE 1, 1: INPUT "DATA FILE NAME "; N$ DTN$ = NS + ".DAT" CLS : PRINT "DATA FILE TO BE LOADED "; DTN$ OPEN "1", #1, DTN$ FOR PR8 = 1 TO 21 INPUT #1, PR$ PRINT PR$ NEXT PR8 INPUT #1, WAVL8 INPUT #1, SR1 INPUT #1, NS8 INPUT #1, NA8 INPUT #1, DC1 157 300 310 320 330 340 350 400 410 420 430 440 450 500 510 520 530 600 610 620 630 640 650 660 700 800 158 INPUT #1, GAN8 INPUT #1, PMT8 INPUT #1, CT11 INPUT #1, CT41 INPUT #1, MS$ I I = 0 INPUT #1, DAT1(I) DAT1(I) = -.4343 8 LOG(1 - (DAT1(I) / DC1)) I = I + 1 IF EOF(1) THEN 500 ELSE GOTO 410 I PRINT "NUMBER OF SAMPLES BE TAKEN", NS% PRINT "NUMBER OF DATA BE READ", I - 1; CLOSE #1 I LOCATE 25, 1: COLOR 12 PRINT "COMMAND: Plot View Window Kfit Lpt plotiT pfF New Smooth BC Exit"; COLOR 15, 9 KEY$ = INKEYS SELECT CASE KEY$ CASE up", up" GOTO 1000 CASE "V", "VI: GOTO 2000 CASE IIW" I "W" GOTO 3000 CASE "K", uku GOTO 6000 CASE "N", "nu CLOSE #1 GOTO 130 CASE "L", "1" GOTO 4000 CASE "S", "S" GOTO 7000 CASE "B", "b" GOTO 8000 CASE "T", "t" GOTO 9500 CASE "F", ufn GOTO 9000 CASE "E", new SCREEN 0: STOP CASE ELSE END SELECT GOTO 630 END 1000 '*************** PLOT **************** 1010 I 1020 SCREEN 9 1030 COLOR 15, 9 1040 WINDOW (-.05 8 NS8, —.004)-(1.05_8 NS8, .004) 1050 CLS 1060 TM = .001 8 NS8 8 SR 1070 LINE (0, 0)-(NS8, 0), 12, , &HCCCC 1080 LINE (NS8, O)-(NS8, .0002), 12 1090 LINE (NS8 / 2, 0)-(NS8 / 2, .0002), 12 1100 LOCATE 14, 70: PRINT USING "####.### ms"; TM; 1110 LOCATE 14, 36: PRINT USING "####.### ms"; TM / 2; 1200 LINE (0, -.004)—(NS8, -.004), 13, , &H8080 1210 LINE (0, -.002)-(NS8, -.002), 13, , &H8080 1220 LINE (0, .002)-(NS8, .002), 13, , &H8080 1230 LINE (0, .004)-(NS8, .004), 13, , &H8080 1240 LINE (0, .003)-(NS8, .003), 13, , &H8080 1250 LINE (0, .001)-(NS8, .001), 13, , &H8080 1260 LINE (0, -.003)-(NS8, -.003), 13, , &H8080 1270 LINE (0, -.001)-(NS8, -.001), 13, , &H8080 1300 I 1310 IF NS8 > 640 THEN MP8 = INT(NS8 / 640) ELSE MP8 1 1320 FOR I = 0 TO NS8 STEP MP8 1330 PSET (I, DAT1(I)) 1340 NEXT I 1350 I 1360 LOCATE 4, 50 1370 PRINT "WAVELENGTH (nm) "; WAVL8 1380 LOCATE 5, 50 1390 PRINT "SAMPLING RATE (us) "; SR1 1400 LOCATE 6, 50 1410 PRINT "NUMBER OF AVERAGES "; NA8 1420 LOCATE 2, 2: PRINT "0.004" 1430 LOCATE 23, 2: PRINT "-0.004" 1440 CLOSE #1 1450 LKEY$="P" 1500 GOTO 600 1600 I 2000 '************* VIEW DATA *********************** 2010 I 2020 LOCATE 1, 1 2030 INPUT "SELECT VIEW WINDOW [t1,t2] ", X11, X2! 2040 WN1 = X21 - X11 2050 SCREEN 9 2060 COLOR 15, 9 2070 WINDOW ((X11 - .05 8 WN), -.004)-((X21 + .05 8 WN), .004) 2080 CLS 2100 T0 = X1: TM = X2 2110 LINE (X11, 0)-(X21, 0), 12, , &HCCCC 159 160 2120 LINE (x21, O)-(X21, .0002), 12 2130 LINE ((X11 + WN / 2), 0)-((X11 + WN / 2), .0002), 12 2140 LOCATE 14, 70: PRINT USING "####.### ms"; TM; 2150 LOCATE 14, 5: PRINT USING "t =###.### ms"; X1; 2160 LOCATE 14, 36: PRINT USING "####.### ms"; X1 + WN / 2; 2200 LINE (X11, -.005)-(X11, .005) ' 2210 LINE (X11, -.004)-(x21, -.004), 13, , &H8080 2220 LINE (X11, -.002)-(X21, -.002), 13, , &H8080 2230 LINE (x11, .002)-(X21, .002), 13, , &H8080 2240 LINE (X11, .004)—(X21, .004), 13, , &H8080 2250 LINE (X11, .001)-(X21, .001), 13, , &H8080 2260 LINE (x11, .003)-(X21, .003), 13, , &H8080 2270 LINE (X11, -.001)-(x21, -.001), 13, , &H8080 2280 LINE (x11, —.003)-(X21, -.003), 13, , &H8080 2300 I 2400 B8 = 1000 8 X11 / SR1: E8 = 1000 8 X21 / SR1 2410 NSL8 = 1000 8 WN1 / SR1: XINC1 = WN1 / NSL8: XL1 = X11 2420 FOR I = B8 TO E8 2430 PSET (XL1, DAT1(I)) 2440 XL1 = XL1 + XINC1 2450 NEXT I 2500 LOCATE 4, 50 2510 PRINT "WAVELENGTH (nm) "; WAVL8 2520 LOCATE 5, 50 2530 PRINT "SAMPLING RATE (us) "; SR1 2540 LOCATE 6, 50 2550 PRINT "NUMBER OF AVERAGES "; NA8 2560 I 2570 LKEY$="V" 2600 GOTO 600 2700 I 3000 '************** WINDOW ******************** 3010 I 3020 LOCATE 1, 1 3030 INPUT "SELECT WINDOW [tl,t2,V(min),V(max)] ", X11, X21, Y11, Y21 3040 WN1 = X21 - X11: YM1 = Y21 - Y11 3050 SCREEN 9 3060 COLOR 15, 9 3070 WINDOW ((X11 - .05 8 WN), Y11 - .05 8 YM1)-((X21 + .05 8 WN), Y21 + .05 8 YM1) 3080 CLS 3100 T0 = x11: TM = X21 3110 LINE (X11, 0)-(X21, 0), 12, , &HCCCC 3120 LINE (X21, 0)-(X21, .03 8 YM1), 12 3130 LINE ((X11 + WN / 2), 0)-((X11 + WN / 2), .03 8 YM1), 12 3150 IF Y11 < 0 AND Y21 > 0 THEN CSL8 = 1 + Y21 8 25 / YM1 LINE (x11, 0)-(X21, 0), 12, , &HCCCC 161 LINE (X21, 0)—(X21, .03 8 YM1), 12 LINE ((X11 + WN / 2), 0)-((x11 + WN / 2), .03 8 YM1), 12 ELSE CSL8 = 23 YL1 = Y11 + .01 8 YM1 LINE (X11, YL1)-(X21, YL1), 12, , &HCCCC LINE (x21, YL1)-(X21, YL1 + .05 8 YM1), 12 LINE ((X11 + WN / 2), YL1)-((X11 + WN / 2), YL1 + .05 8 YM1), 12 3160 END IF 3170 LOCATE CSL8, 70: PRINT USING "####.### ms"; TM; 3180 LOCATE CSL8, 1: PRINT USING "####.### ms"; x1; 3190 LOCATE CSL8, 36: PRINT USING "####.### ms"; X1 + WN /2; 3200 LINE (X11, Y11)-(X11, Y21) 3210 LINE (X11, .001)—(X21, .001), 13, , &H8080 3220 LINE (X11, .002)-(X21, .002), 13, , &H8080 3230 LINE (X11, .003)-(X21, .003), 13, , &H8080 3240 LINE (X11, .004)-(X21, .004), 13, , &H8080 3250 LINE (X11, .005)-(X21, .005), 13, , &H8080 3260 LINE (X11, .006)-(X21, .006), 13, , &H8080 3270 LINE (X11, -.001)-(X2!, -.001), 13, , &H8080 3280 LINE (X11, -.002)-(X21, -.002), 13, , &H8080 3290 LINE (X11, -.003)-(X21, -.003), 13, , &H8080 3300 LINE (X11, -.004)-(X21, -.004), 13, , &H8080 3310 LINE (X11, -.005)-(X21, -.005), 13, , &H8080 3320 LINE (X11, -.006)-(x21, -.006), 13, , &H8080 3400 I 3500 B8 = 1000 8 X11 / SR1 3510 E8 = 1000 8 X21 / SR1 3520 NSL8 = 1000 8 WN1 / SR1 3530 XINC1 = WN1 / NSL8 3540 XL1 = X11 3550 FOR I = 88 TO E8 3560 PSET (XL1, DAT1(I)) 3570 XL1 = XL1 + XINC1 3580 NEXT I 3590 I 3600 LOCATE 3, 5 3610 PRINT USING " +##.### "; Y21; 3620 LOCATE 23, 5 3630 PRINT USING " +##.### "; Y11; 3640 LOCATE 4, 50 3650 PRINT "WAVELENGTH (nm) "; WAVL8 3660 LOCATE 5, 50 3670 PRINT "SAMPLING RATE (us) "; SR1 3680 LOCATE 6, 50 3690 PRINT "NUMBER OF AVERAGES "; NA8 3700 I 3750 LKEY$="W" 162 3800 GOTO 600 3900 I 4000 '*************** PRINT OUT ******************* 4010 I 4020 SELECT CASE LKEYs CASE "P" GOTO 4500 CASE "W" GOTO 5000 CASE IIVII GOTO 5500 CASE ELSE PRINT "NON AVAILABLE FUNCTION" 4030 END SELECT 4100 I 4500 I ------------- PRINT PLOT ----------- 4510 I 4520 SCREEN 2 4530 WINDOW (-.05 8 NS8, -.005)-(1.05 8 NS8, .005) 4540 CLS 4550 TM = .001 8 NS8 8 SR 4560 LINE (0, 0)-(NS8, 0), 7, , &HAAAA 4570 LINE (NS8, 0)-(NS8, .0005), 7 4580 LINE (NS8 / 2, 0)-(NS8 / 2, .0005), 7 4590 LOCATE 14, 70: PRINT USING "####.### ms"; TM; 4600 LOCATE 14, 36: PRINT USING "####.### ms"; TM / 2; 4610 LINE (0, —.005)-(0, .005) 4620 LINE (.12 8 NS8, .002)-(NS8, .002), 7, , &H8080 4630 LINE (.12 8 NS8, .004)-(NS8, .004), 7, , &H8080 4640 LINE (.12 8 NS8, -.002)-(NS8, -.002), 7, , &H8080 4650 LINE ( 12 8 NS8, -.004)-(NS8, -.004), 7, , &H8080 4660 I 4700 IF NS8 > 640 THEN MP8 = INT(NS8 / 640) ELSE MP8 = 1 4710 FOR I = 0 TO NS8 STEP MP8 4720 PSET (I, DAT1(I)) 4730 NEXT I 4740 I 4750 LOCATE 4, 50 4760 PRINT "WAVELENGTH (nm) "; WAVL8 4770 LOCATE 5, 50 4780 PRINT "SAMPLING RATE (us) "; SR1 4790 LOCATE 6, 50 4800 PRINT "NUMBER OF AVERAGES "; NA8 4810 CLOSE #1 4820 I 4830 FOR I = 1 TO 17 4840 LPRINT CHR$(10) 4850 NEXT I 4860 LPRINT "DATA FILE NAME "; NS 4870 LPRINT "" 4880 4881 4882 4883 4884 4885 4886 4887 4888 4890 4900 4910 4920 4930 4940 4950 5000 5010 5020 5030 5040 5050 5060 5070 5080 5090 5100 5110 5120 5130 5140 5141 .05 8 YM!), 163 LPRINT "WAVELENGTH (nm) "; WAVL% LPRINT "SAMPLING RATE (us) "; SR! LPRINT "NUMBER OF SAMPLES "; NS% LPRINT "NUMBER OF AVERAGES "; NA% LPRINT "DC OFFSET (v) "; DC! LPRINT "GAIN OF PRE-ADC AMP "; GAN% LPRINT "PMT VOLTAGE (v) "; PMT% LPRINT "TIME OF TRIGGERING THE LASER (ms) "; CT11 LPRINT "TIME DELAY OF COUNTER 4 (ms) "; CT4! LPRINT CHR$(12) SHELL "GRAPHICS" LP$ = INKEY$ SELECT CASE LP$ CASE "R", urn LPRINT CHR$(12) SCREEN 9 GOTO 1000 CASE ELSE END SELECT GOTO 4910 I I -------------- PRINT WINDOW ------------------ I SCREEN 2 WINDOW ((X11 - .05 8 WN), Y1! - .05 8 YM!)-((X2! + .05 8 WN), Y2! + .05 8 YM!) CLS T0 = x1!: TM = x2! LINE (x11, 0)-(x2:, 0), 12, , &HAAAA LINE (x21, 0)—(x21, .03 8 YM1), 12 LINE ((X1! + WN/2), 0)-((x1! + WN / 2), .03 8 YM!),12 IF Y1! < 0 AND Y2! > 0 THEN CSL% = 1 + Y2! 8 25 / YM1 LINE (x11, 0)-(x21, 0), 12, , &HAAAA LINE (x21, 0)-(x21, .03 8 YM1), 12 LINE ((X11 + WN / 2), 0)-((x1! + WN / 2), .03 8 MI), 12 ELSE CSL% = 23 YL! = Y1! + .01 8 YM! LINE (x11, YL!)-(X2!, YL1), 12, , &HAAAA LINE (x21, YL!)-(X2!, YL! + .05 8 YM!), 12 LINE ((X11 + WN / 2), YL!)-((X1! + WN / 2), YL! + 12 END IF LOCATE CSL%, 70: PRINT USING "####.### ms"; TM; LOCATE CSL%, 1: PRINT USING "####.### ms"; x1; LOCATE CSL%, 36: PRINT USING "####.### ms"; x1 + WN/2; LINE (x11, Y1!)-(X1!, Y2!) LINE (x11, .001)-(x2!, .001), 9, , &H8080 5142 5143 5144 5145 5146 5147 5148 5149 5150 5151 5152 5160 5170 5180 5190 5200 5210 5220 5230 5240 5250 5260 5261 5262 5263 5264 5265 5266 5267 5268 5269 5270 5280 5290 5300 5301 5302 5303 5304 5305 5306 5307 5308 5309 5310 5311 5320 5330 5340 5350 XINC! XL! = (X1:, (X1!, (X1!, (X1!, (X1!, (X1:, (X1:, (X11, (X1:, (X1!, (X1:, 1000 * X1! 1000 * X2! = 1000 = WN! X1! 8 WN! / NSL% .002)-(x2!, .003)-(x2:, .004)-(x21, .005)-(x2!, .006)-(X2!, -.001)-(X2!, -.002)-(x2!, -.003)-(x2!, -.004)-(x2!, -.005)-(x2!, -.006)-(X2!, FOR I = 8% TO E% PSET (XL!, XL! = NEXT I LOCATE PRINT USING " +##.### "; Y2!; LOCATE PRINT USING " +##.### "; LOCATE PRINT XL! 3, 23, 4: DAT1(I)) + XINC! "WAVELENGTH LOCATE 5, 50 / SR! / SR! / SR! 164 .002), .003), .004), .005), .006), -.001), -.002), -.003), -.004), -.005), -.006), (um) PRINT "SAMPLING RATE (us) LOCATE 6, 50 "NUMBER OF AVERAGES PRINT CLOSE #1 FOR I = 1 TO 17 CHR$(10) LPRINT NEXT I LPRINT LPRINT LPRINT LPRINT LPRINT LPRINT LPRINT LPRINT LPRINT LPRINT LPRINT LPRINT SHELL "DATA FILE NAME "WAVELENGTH "SAMPLING RATE (hm) (US) "NUMBER OF SAMPLES "NUMBER OF AVERAGES II DC "GAIN OF PRE-ADC AMP OFFSET "PMT VOLTAGE "TIME OF TRIGGERING THE LASER "TIME DELAY OF COUNTER 4 CHR$(12) "GRAPHICS" LP$ = INKEYS SELECT CASE LP$ (V) (V) Y11; I SD\O\D\O\D I I I I I N$ &H8080 &H8080 &H8080 &H8080 &H8080 &H8080 &H8080 &H8080 &H8080 &H8080 &H8080 “‘§“““‘ \“‘\‘ 000000- WAVL% SR! NA% " ; WAVL% "; SR! "; NS% "; NA% "; DC! "; GAN% "; PMT% (m8) (m8) "0 I II. I CTl! CT4! 5360 5370 5400 5500 5510 5520 5530 5540 5550 5560 5570 5580 5590 5600 5610 5611 5612 5613 5614 5615 5620 5630 5640 5650 5660 5670 5680 5690 5700 5710 5720 5730 5740 5750 5760 5770 5780 5790 5800 5801 5802 5803 5804 5805 5806 165 CASE IIRII ' IIrII LPRINT CHR$(12) SCREEN 9 GOTO 3060 CASE ELSE END SELECT GOTO 5340 I I ------------- PRINT VIEW ---------------- I SCREEN 2 WINDOW ((Xl! - .05 8 WN), -.74)-((x2: + .05 8 WN), .8) CLS T0 = X1: TM = X2 LINE (X11, 0)-(X2!, 0), 12, , &HAAAA LINE (X2!, 0)-(x2!, .0004), 12 LINE ((Xl! + WN / 2), 0)-((X1! + WN / 2), .0004), 12 LOCATE 14, 70: PRINT USING "####.### ms"; TM; LOCATE 14, 5: PRINT USING "t =###.### ms"; X1; LOCATE 14, 36: PRINT USING "####.### ms"; X1 + WN / 2; LINE (X1!, -.004)-(X1!, .004) LINE ((Xl! + .12 8 WN), .002)-(X2!, .002), 9, , &H8080 LINE ((Xl! + .12 8 WN), .004)-(X21, .004), 9, , &H8080 LINE ((X1! + .12 8 WN), -.002)-(X2!, -.002),9, ,&H8080 LINE ((X11 + .12 8 WN), -.OO4)-(X2!, -.004),9, ,&H8080 I B8 = 1000 8 X1! / SR!: E8 = 1000 8 X2! / SR! NSL8 = 1000 8 WN! / SR!: XINC! = WN! / NSL8: XL! = X1! FOR I = B8 TO E8 PSET (XL!, DAT!(I)) XL! = XL! + XINC! NEXT I LOCATE 4, 50 PRINT "WAVELENGTH (nm) "; WAVL8 LOCATE 5, 50 PRINT "SAMPLING RATE (us) "; SR! LOCATE 6, 50 PRINT "NUMBER OF AVERAGES "; NA8 CLOSE #1 FOR I = 1 TO 17 LPRINT CHR$(10) NEXT I LPRINT "DATA FILE NAME "; N$ LPRINT "" LPRINT "WAVELENGTH (nm) "; WAVL8 LPRINT "SAMPLING RATE (us) "- SR! LPRINT "NUMBER OF SAMPLES "; NS8 LPRINT "NUMBER OF AVERAGES "; NA8 LPRINT "DC OFFSET (v) "; DC! LPRINT "GAIN OF PRE-ADC AMP "; GAN8 5807 5808 5809 5810 5820 5830 5840 5850 5860 5900 6000 6010 6020 6030 6040 6050 6060 6070 6080 6090 6100 6110 6120 166 "PMT VOLTAGE (v) PMT8 "TIME OF TRIGGERING THE LASER "TIME DELAY OF COUNTER 4 LPRINT CHR$(12) SHELL "GRAPHICS" LP$ = INKEY$ SELECT CASE LP$ CASE "R", urn LPRINT CHR$(12) SCREEN 9 GOTO 2040 CASE ELSE END SELECT GOTO 5830 I LPRINT LPRINT LPRINT II; CTl! CT4! (ms) "; (ms) "; I************ KFIT FILE OUTPUT ***************** I LOCATE 3, 3 PRINT " COMMENT ? BDP8 1000 8 X1! / SR!: NDP8 EDP8 - BDP8 + 1 KFN$ NS + ".FIT" KDPM! = NDP8 / 1001 SELECT CASE KDPM! CASE 0 TO 1 KMULT8 = 1 CASE 1.0009 TO 2 KMULT8 = 2 CASE 2.0009 TO 3 KMULT8 = 3 CASE 3.0009 T0 4 KMULT8 = 4 CASE 4.0009 TO 5 KMULT8 = 5 CASE 5.0009 TO 6 KMULT8 = 6 CASE 6.0009 TO 7 KMULT8 = 7 CASE 7.0009 TO 8 KMULT8 = 8 CASE 8.0009 TO 9 KMULT8 = 9 CASE 9.0009 TO KMULT8 = 10 CASE ELSE PRINT " END SELECT I OPEN "0", PRINT #3, ": INPUT ; COMT$ 1000 * X2! II II I EDP8 = / SR! 10 TOO MANY DATA POINTS TO FIT ": GOTO 600 #3, KFN$ COMT$ 6130 6140 6150 6160 6170 6180 6190 6200 6300 7000 7010 7020 7030 7040 7050 7060 7070 7080 7090 7100 7110 7120 7130 7140 7150 7160 7170 7180 7190 7200 7210 7220 7230 7240 7250 7260 7270 7300 7400 7500 8000 8010 8120 8130 8140 8150 8200 8210 8230 8300 167 PRINT #3, "ABSORBANCE" PRINT #3, INT(NDP% / KMULT%) PRINT #3, (SR! / 1000000) 8 KMULT8 FOR I = BDP8 TO EDP8 STEP KMULT8 PRINT #3, DAT!(I) NEXT I CLOSE #3 GOTO 600 I '***************** SMOTHING *************** I BDP% = 1000 * X1! / SR!: EDP% = 1000 * X2! / SR! NDP% = EDP% - BDP% + 1 LOCATE 1, 2 INPUT " ENTER THE SMOTHING WINDOW "; SMW8 SMWM8 = (SMW8 - 1) / 2 FOR I = BDP8 TO (BDP8 + SMWM8) FOR N = (I + 1) TO (I + SMWM8) DAT!(I) = DAT!(I) + DAT!(N) NEXT N DAT!(I) = DAT!(I) / (SMWM8 + 1) NEXT I FOR I = (BDP8 + (SMWM8 + 1)) TO (EDP8 - (SMWM8 + 1)) FOR N = (I - SMWM8) TO (I — 1) DAT!(I) = DAT!(I) + DAT!(N) NEXT N FOR N = (I + 1) TO (I + SMWM8) DAT!(I) = DAT!(I) + DAT!(N) NEXT N DAT!(I) = DAT!(I) / SMW8 NEXT I FOR I = (EDP8 - SMWM8) TO EDP8 FOR N = (I - SMWM8) TO (I - 1) DAT!(I) = DAT!(I) + DAT!(N) NEXT N DAT!(I) = DAT!(I) / (SMWM8 + 1) NEXT I PRINT " ****** DONE ****** " GOTO 600 I I************** BACKGROUND CORRECTION *********** I LOCATE 1, 1: PRINT " " LOCATE 1, 1: INPUT "BACKGROUND DATA FILE NAME "; B$ BKN$ = as + ".DAT" OPEN "I", #2, BKN$ FOR PR% = 1 TO 21 INPUT #2, PR$ NEXT PR% INPUT #2, WAVL8 8310 8320 8330 8340 8350 8360 8370 8380 8390 8400 8500 8510 8520 8530 8540 8550 8600 8700 8800 8900 9000 9010 9020 9030 9040 9050 9060 9070 9080 9090 9100 9200 9300 9400 9500 9510 9520 9530 9540 9550 9560 9570 9580 9600 9700 9800 168 SR! NS% NA% DC! GAN% PMT% CTl! CT4! MS$ INPUT INPUT INPUT INPUT INPUT INPUT INPUT INPUT INPUT I #2, #2, #2, #2, #2, #2, #2, #2, #2, I = 0 INPUT #2, BDAT!(I) BDAT!(I) = LOG(1 + (BDAT!(I) / DC!)) DAT!(I) = DAT!(I) - BDAT!(I) I = I + 1 IF EOF(2) THEN 8600 ELSE GOTO 8510 CLOSE #2 PRINT " GOTO 600 I *********** DONE *********** u I************** WRITE PFT DATA FILE **************** LOCATE 1, 1 STN$ = NS + ".SET" PRINT "DATA FILE NAME TO BE SENT ", OPEN "0", #1, STN$ FOR I = 0 TO NS% X = CSNG(SR! * I): Y = PRINT #1, "RD"; PRINT #1, X, Y NEXT I CLOSE #1 PRINT " GOTO 600 I I************** WRITE PLOTIT DATA FILE **************** LOCATE 1, 1 STN$ 1000 8 CSNG(DAT(I)) *********** *************** " DONE STN$ = N$ + ".SET" PRINT "DATA FILE NAME TO BE SENT ", STN$ OPEN "0", #1, STN$ FOR I = 0 TO NS% X = CSNG(SR! 8 I): Y = 1000 8 CSNG(DAT(I)) PRINT #1, X, Y NEXT I CLOSE #1 PRINT " GOTO 600 *********** *************** " DONE "I7'!!!!!!!!!!!!!!T