REDu .‘COUSULF‘ITE‘? STUDIES ON E AND THE ACTIVATIONOE SELENATE LEW? E? av; msr masu green: Dissertation in»; 3n a umvmsm , H" GREGORY LEE 01L. r ithe‘i-De’ ~ 1975 ECHIGAN STAT M . ., This is to certify that the thesis entitled STUDIES ON E. COLI SULFITE REDUCTASE AND THE ACTIVATION 0F SELENATE BY YEAST ATP SULFURYLASE presented by GREGORY LEE DILWORTH has been accepted towards fulfillment of the requirements for ”In ‘\ .a \I 3 3“ I I. \2 (“,ng IIAMVI VIII I a)“ I I \I II—I \\ 'I Ila C142“ I IA 7 Major professor Date OC&O\)'IFZ ICI?IJ’ am - HEW I ”moans. I, ABSTRACT STUDIES ON E. COLI SULFITE REDUCTASE AND THE ACTIVATION OF SELENATE BY YEAST ATP SULFURYLASE By Gregory Lee Dilworth Part I. Studies on E. coli Sulfite Reductase The six electron reduction of sulfite to sulfide catalyzed by g. 32;; sulfite reductase was studied. Two approaches were used to elucidate the intermediate forms of sulfur that occur during the reduction. The first series of experiments involved the binding of radioactive sulfur to the purified enzyme under conditions that might lead to partially reduced intermediates. Reduced nicotin— amide adenine dinucleotide phosphate-dependent binding of radioactive sulfur from sulfite could be observed under these conditions. This binding was found to be inversely proportional to the specific activity of the homogeneous enzyme preparations. Using published values as the maxi- mum specific activity possible, it could be calculated that there would be no binding if the preparation was completely active. If the preparation was completely in- active, approximately one mole of sulfur would be bound at saturation to one mole of enzyme. The inactivation, Gregory Lee Dilworth which permitted increased binding, occurred during purifica- tion and long term storage of the preparation. The radio- active sulfur appeared to be covalently bound as the radio- activity could not be separated from the enzyme by extensive dialysis, gel filtration, and treatment with sodium dodecyl sulfate or ethanol. The binding was concluded to be associ— ated with an inactivation event as opposed to the formation of a bound intermediate. The second approach was an attempt to modify the enzyme such that the enzyme catalyzed only a partial reduction. A number of treatments were used, but the reaction catalyzed could not be altered. These studies did, however, result in the finding that sodium formaldehyde sulfoxylate is a good inhibitor of sulfite reductase. Part II. The Activation of Selenate by Yeast ATP Sulfurylase Crude yeast extracts containing sulfate activating enzymes, were found to catalyze an adenosine 5'-triphosphate (ATP) and cysteine-dependent reduction of selenate to elemental selenium. The requirement for ATP implied the formation of adenosine phosphoselenate (APSe) analogous to the formation of adenosine phosphosulfate (APS). The formation of a selenium compound with the electrophoretic and stability properties of APS could be detected. However, an ion-exchange chromatographic separation, which required more time than an electrophoretic separation, failed to Gregory Lee Dilworth resolve an adenine-containing selenium compound. A direct spectrophotometric procedure was developed to assay the ATP, ATP sulfurylase, glutathione, and pyrophosphatase— dependent formation of elemental selenium from selenate. The enzyme—catalyzed formation of elemental selenium showed the same unusual kinetics and glutathione dependency as the chemical reduction of selenite to elemental selenium by glutathione. It was shown that two phosphates were releas- ed for each "active" selenium formed. The observed reac- tivity towards thiols and instability properties of the enzymatic product were found to be those predicted for selenium anhydrides. By analogy to sulfur chemistry, the product of the thiolytic cleavage of a selenium anhydride would be easily converted to selenite. The selenite would then be reduced by the thiol to elemental selenium. Thus it was concluded that ATP sulfurylase can catalyze the formation of APSe, or a similar selenium anhydride. The anhydride can be reduced by thiol compounds in a manner similar to the reduction of selenite by thiols. The role of ATP sulfurylase in the in 3119 reduction of selenate is discussed. STUDIES ON E. COLI SULFITE REDUCTASE AND THE ACTIVATION OF SELENATE BY YEAST ATP SULFURYLASE By Gregory Lee Dilworth A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHYLOSOPHY Department of Botany and Plant Pathology 1975 To my family ii ACKNOWLEDGMENTS I would like to thank Dr. Robert S. Bandurski for his guidance and his tolerance towards me during my graduate career at Michigan State University. The patience and assistance of my graduate committee; Dr. D. Delmer, Dr. A. Kivilaan, and Dr. L. Wilson is gratefully acknowledged. I would particularly like to thank Dr. L. Wilson and Dr. G. Kennedy for providing many insights into the biochemistry of sulfur. I would also like to thank Dr. A. Ehmann for his critical reading of this thesis. The financial support from the National Science Foundation (GB-18353—X and GB—UOSZi—X) and a graduate teaching assistantship from the Department of Botany and Plant Pathology is gratefully acknowledged. iii TABLE OF CONTENTS Chapter LIST OF TABLES - — — - — — _ _ _ _ _ _ LIST OF FIGURES - - - - — — _ _ - _ _ _ LIST OF ABBREVIATIONS - - - — — _ _ _ _ _ INTRODUCTION Sulfur Metabolism - - - — — _ _ - _ _ Selenium Metabolism — - — - - — — _ _ Part I. STUDIES ON E. COLI SULFITE REDUCTASE LITERATURE REVIEW Role of Sulfite Reductase in the Assimilatory Sulfate Reduction Pathway - — — - _ _ _ Properties of Sulfite Reductase from Various Organisms — - - — — _ _ _ _ _ _ _ The Mechanism of the Enzymatically Catalyzed Reduction of Sulfite — - — - — - MATERIALS AND METHODS Chemicals - - - — — _ — _ _ - _ _ E. 221; Sulfite Reductase Preparations - - - Sulfite Reductase Assays - - — — _ - _ Protein Assays - — — - — — _ _ _ - _ Gel Electrophoresis - — — - — _ _ _ _ 353 Binding Assay _ - - _ _ - _ - _ _ Stoichiometry and Inhibition Assays — - — - iv Page vii viii 12 15 15 18 20 20 21 22 Chaper Page RESULTS Enzyme Purification — — — - - - - - - 2# Gel Electrophoresis of Purified Enzyme - — — 24 358 Binding Studies - - - — — — - - - 28 Stoichiometry and Inhibition Studies - — - 38 DISCUSSION - - - - - - - - — - - - - - 41 Part II. THE ACTIVATION OF SELENATE BY YEAST ATP SULFURYLASE LITERATURE REVIEW The Biology of Selenium - - - - - - - - 47 The Relationship between the Biology of Selenium and Sulfur - - - - - - - - - 49 Selenium Metabolism — — - — - - - - - 53 The Relationship between the Metabolism of Selenium and Sulfur - — - - - - — - - 58 MATERIALS AND METHODS Chemicals - - — — — _ _ _ - _ _ _ 63 Enzyme Preparations - - - - - - - - - 64 Elemental Selenium Filtration Assay - — - - 67 Electrophoresis - — - - - — - - - — 68 Column Chromatography - - - - - - - - 68 Direct Spectrophotometric Assay of Elemental Selenium — — - - — — — _ _ _ - . _ 7O Selenate—dependent phosphate release from ATP- 70 RESULTS Elemental Selenium Formation from Selenate - 72 Studies on APSe and PAPSe Formation - - - - 74 V Chapter Page Electrophoretic Analysis of Enzymatic Products - - - — 77 Column Chromatographic Analysis of Enzymatic Products — - — - - — - - 77 Direct Spectrophotometric Assay - — - - — 91 Requirements and Stoichiometry of the ATP Sulfurylase-catalyzed Reaction — - - - 92 Stability of the Enzymatic Product — — — — 98 DISCUSSION — - - — — - - — — - — — - 1ou APPENDIX. SULFITE- DEPENDENT NADPH OXIDASE ACTIVITY IN CULTURED TOBACCO CELL EXTRACTS - — - — - — — 115 BIBLIOGRAPHY — — - — - - — — — — - - - 131 vi 10. LIST OF TABLES Properties of the purified sulfite reductase preparations - - — - — - _ _ - _ Purification of sulfite reductase — - - — Dialysis experiments using pretreated membranes - - - — — — _ _ - _ _ - Inhibitor studies on sulfite reductase - — The effect of cysteine, ATP, and enzyme on elemental selenium formation - - - - - - Influence of NADPH on elemental selenium formation - - - — — — — — _ _ _ _ The activation of dialyzed tobacco extract by boiled tobacco extract — - - — _ _ _ The disappearance of sulfite in the reaction mixtures - - - - — _ - _ _ _ _ - Boiled enzyme factor consumption during the reaction - - - - - - - - _ - _ Effect of various additions on NADPH oxidase activity - — - — — - _ - _ - _ _ vii Page 11 25 29 39 75 76 119 122 124 127 LIST OF FIGURES Figure Page 1. The proposed pathway of assimilatory sulfate reduction in microorganisms - - - - 2 2. Gel electrophoresis studies of sulfite reductase - — — - - — — _ _ _ _ _ _ 26 3. NADPH dependent binding at various substrate concentrations - - - - — — — _ _ _ 31 4. Saturation of NADPHvdependent binding - - — - 3LI 5. Saturable binding as a function of enzyme inactivation - — - - — - - - — — — - 35 6. Elution of bound radioactive sulfur with protein and sulfite reductase activity during Sepharose 6B column chromatography - - — - - 37 7. Standard curve for selenite assay - - - - - 73 8. The enzymatic production of a radioactive selenium peak with the same Rf as APS during paper electrophoresis - - - - 79 9. The acid lability of a radioactive selenium peak with the same Rf as APS during paper electrophoresis - — - - - - 81 10. Standard curve for organic phosphate determination - - — - - - - - - - - - 83 11. A. Enzymatic PAPS production as resolved by DEAE Sephadex-A 25 column chromatography - - - 85 B. Enzymatic production of selenate metabolites by the sulfate activating enzymes as resolved by DEAE Sephadex-A 25 column chromatography - - - - - - - 85 12. Column chromatography of the products formed from cultured tobacco cell extracts with sulfate and selenate - - - - — - — - - 88 viii Figure 13. 14. 15. 16. 17. 18. 19. 20. 21. Column chromatography of the product formed by yeast ATP sulfurylase with sulfate and selenate Optical density at 380 nm of various concentrations of sodium selenite in 10 mM reduced glutathione — - - - — - - - — The ATP sulfurylase-dependent increse in the optical density at 380 nm with time at various reduced glutathione concentrations _ _ _ _ The effect of various reaction components on the production of elemental selenium (selenite equivalents) by ATP sulfurylase — — Ratio of phosphate and elemental selenium (selenite equivalents) formation with time - Stability ofthe enzymatic product _ - _ _ Proposed pathway for the ATP sulfurylase— catalyzed reduction of selenate — - - - - pH optima for tobacco sulfite-dependent NADPH oxidase activity —- — — — — - — — Oxygen consumpt mixtures ion with various reaction ix Page 90 93 95 97 100 103 112 121 125 LIST OF ABBREVIATIONS ADP Adenosine 5'-diphosphate AMP _ Adenosine 5'-phosphate 3'-AMP = Adenosine 3'-phosphate APS = Adenosine 5'-phosphosulfate APSe = Adenosine 5'—phosphoselenate ATP = Adenosine 5'-triphosphate BSA = Bovine Serum albumin DEAE Diethylaminoethyl- EDTA = Ethylenediamine tetraacetate FAD Flavin adenine dinucleotide FMN _ Flavin mononucleotide Glucose-6-P = Glucose-6-phosphate GSSG = Oxidized glutathione GS-Se-SG = Selenodiglutathione GS-SeH = Glutathione selenopersulfide GSH = Reduced glutathione K-phosphate = Potassium phosphate MV = Methyl viologen NADP+ = Nicotinamide adenine dinucleotide phosphate NADPH = Reduced nicotinamide adenine dinucleotide phosphate PAPS = 3'-phosphoadenine-5'-phosphosulfate PAPSe = 3'-phosphoadenine-5'-phosphose1enate Pi = Inorganic phosphate PPi : Inorganic pyrophosphate SDS = Sodium dodecyl sulfate Tris = 2-amino-2-hydroxymethyl-1,3-propanediole Vol = Volume Wt 2 Weight III INTRODUCTION This dissertation presents research on enzyme-catalyzed reactions involved in the metabolism of inorganic compounds of sulfur and selenium. Sulfur Metabolism Inorganic sulfate can be utilized for the synthesis of organic sulfur compounds by microorganisms and plants, or as the terminal electron acceptor during anaerobic respiration by some bacteria. The first process is called assimilatory sulfate reduction, while the second is termed dissimilatory sulfate reduction. The research presented here was performed on the assimilatory sulfite reductase of Escherichia coli. The overall assimilatroy sulfate reduction pathway, as it is thought to occur in microorganisms (1), is diagramed in Figure 1. The sulfur passes sequentially through the enzymatic reaction steps catalyzed by ATP sulfurylase (E.C. 2.7.7.4), APS kinase (E.C. 2.7.1.25), the "PAPS reductase system" (which includes enzyme A, enzyme B, and fraction C), sulfite reductase (E.C. 1.8.1.2) and O-acetylserine sulfhydrolase (E.C. 4.2.99.8). .ncaanoo * .AHV hosmam one Sowafiz Scam dcpmmv< .wsmaqmeOOHoHs SH Soaposccp commHSm kHOpmHaEHmmm mo magneto Ucmoaopg 639 .H mazwam i N mmfisnwoxmogaa 225mg: mzzmm Ema 1:83 192 m 1.2,. m an E + + M n n54.” n< M n< Mamas W $589158 $-26 ”1253. M853 $5 ”1ng we mmz was; .5 "=8 .3 “$8 mzlmmaEero WEBB m 3 // is .Qmmum NE 9 .bé >643 888 Nave mos; E92 + < sz Inorganic sulfate is relatively non-reactive and requires activation before other reactions can occur. This activation, which consumes three "high—energy" phosphate bonds, is catalyzed by the enzymes ATP sulfurylase and APS kinase. After being activated the sulfate can be directly incorporated into organic sulfate esters and sulfonic acids, or the activated sulfate can be enzymatically reduced by the "PAPS reductase system" and sulfite reductase to a form that can be incorporated into the sulfur amino acids. The pathway presented in Figure 1 is a hypothesis that is consistent with the presently available evidence. The mechanism of the two electron transfer to sulfate by the reduced carrier and the six electron reduction catalyzed by sulfite reductase are unknown. The mechanism of the sulfite reductase catalyzed reaction was the area investigated by the studies presented in the first part of this dissertation. Information about the chemical transformations that occur during the enzyme-catalyzed reduction of sulfite to sulfide was sought by attempting to form and detect inter— mediates that were only partially reduced. Two different experimental approaches were used. The first approach involved studying the binding of radioactive sulfur, from radioactive sulfite, to the enzyme under incubation con— ditions that had the potential of trapping partially reduced intermediates. The second approach was an attempt to modify the enzyme such that it would catalyze only a partial reduction. Characterization of the intermediates formed would have provided information about the chemical conver- sions taking place during the reaction. Unfortunately, this characterization was not possible as neither approach gave results indicating the formation of intermediate_products. The binding experiments showed that under the proper conditions radioactive sulfur could be bound to the enzyme. However, the binding was concluded to be a phenomenon asso- ciated with an inactivation process as opposed to the trap— ing of an intermediate. The attempts to modify the enzyme activity provided information about sulfite reductase inhib- itors, but did not change the reaction catalyzed. Selenium Metabolism The element selenium was discovered by Berzelius in 1817. It was considered a chemical oddity until the early 1930's, when its role as the toxic agent in certain range plants was described. Further studies have since been con- ducted on the toxicology of selenium along with nutritional investigations. The nutritional studies were prompted by‘ the recOgnition that selenium is an essential nutrient for animals and microorganisms. Early studies on the toxicological properties of selenium compounds revealed the interaction in biological organisms between selenium and another group VIA element, sulfur. Several lines of evidence have evolved since this time that demonstrate that the metabolism of selenium at least parallels the metabolism of sulfur. This conclusion is based on the following evidence: (a) the antagonism of sulfur compounds towards the toxic effects of selenium compounds, (b) the similarity between naturally occurring selenium compounds and the naturally occurring sulfur compounds, (0) the approximately equal distribution of selenium and sulfur in biological organisms, (d) the ability of several enzymes that normally catalyze reactions involv- ing sulfur compounds to metabolize the analogous selenium compounds. To date, only one of the enzymes involved in the assimilatory sulfate reduction pathway has been shown to effectively utilize an analogous selenium compound as a substrate. This enzyme, ATP sulfurylase, catalyzes the first reaction of the pathway, the activation of sulfate. Wilson and Bandurski (2) showed that selenate could act as an effective substrate for ATP sulfurylase and provided evidence for the formation of APSe. Recently, another laboratory has reported (3) that the formation of APSe could in fact not be demonstrated using techniques similar to those used by Wilson and Bandurski. The purpose of the second part of this thesis was to investigate the activation 0f selenate by ATP sulfurylase. It was concluded that APSe, Or a similar selenate anhydride was indeed formed during the reaction. A mechanism for the in vitro reduction of selenate to elemental selenium is proposed and its potential relation— Ship to the in vivo pathway is discussed. Part I. STUDIES ONfiE. COLI SULFITE REDUCTASE LITERATURE REVIEW The ability to reduce sulfite to sulfide has been found in most microorganisms and plants. Since the early 1950's, several laboratories have spent considerable effort in studying this reduction and how it relates to the over- all biological assimilation of sulfur. Several review articles containing discussions on this reduction have been published (4—10). Role of Sulfite Reductase in the Assimilatory Sulfate Reduction Pathway The involvment of a reductive step from sulfite to sulfide in the pathway of biological sulfate reduction was shown by four experimental approaches. Enzyme, mutant, in 1112 labeling and regulation studies have provided a sound basis for the inclusion of the enzyme, sulfite reductase (E.C. 1.8.1.2. and E.C. 1.8.99.1), which catalyzes the re— duction of sulfite to sulfide in the pathway of assimila— tory sulfate reduction. 7 The ability of sulfite to be metabolized has been shown by numerous studies with microorganisms. These studies dem— onstrated that sulfite can be utilized by these organisms as their sole, nutritionally required, sulfur source (see reference 6). The relationship between the metabolism of sulfite and the metabolism of sulfate can be shown by compe— tition studies. These studies showed that the metabolism of radioactive sulfate was suppressed by adding non-radioactive sulfite. The unlabeled sulfite had mixed with the labeled sulfite intermediate thus diluting the amount of radioactive sulfur appearing as reduced organic compounds. These Stud- ies were performed with E. ggli (11), Salmgnglla (12), and mung bean leaves (13). The same kind of study using sulfide has shown that sulfide likewise suppresses the reduction and incorporation of radioactive sulfate (14). The demonstra— tion of the formation of sulfite from sulfate in tomato plants (15), tobacco leaves (16), mung bean leaves (17), and yeast (18), further implicates the role of sulfite as an intermediate in the reduction of sulfate. Enzymological studies have shown that in 31339 the en- zyme sulfite reductase can catalyze the reduction of sulfite to sulfide. The occurrence of another enzyme, 0- acetylserine sulfhydrolase which forms cysteine from hydro- gen sulfide and O-acetylserine provides a potential pathway for the reduction and incorporation of sulfite into amino acids. The formation of free sulfite from an enzymatic system has been shown (19), but further studies have sug— gested that the product is a "bound" sulfite (20) which can 8 be liberated by reaction with reduced thiol compounds (21). The existence of enzymes which are capable of catalyzing the proposed reaction, forming the substrate, and utilizing the product also provides evidence for this pathway. A number of studies have been performed with genetic mutations in the sulfate reduction pathway. Several of these mutations have been shown to be associated with sul- fite reductase activity. Mutations which do not permit growth on sulfate or sulfite but do permit growth on sulfide or sulfur amino acids have been shown in Salmonella (22), yeast (23), Neurospora crassa (24), and Aspergillgs nidulans (25) to be associated with the lack of detectable sulfite reductase activity. Thus the genetic evidence also supports the concept that sulfite reductase is an essential part of the sulfate assimilatory pathway. The regulation of sulfite reductase also supports this concept. The level of sulfite reductase has been shown to be regulated by an end—product of the assimilation pathway, cysteine. Cysteine has been shown to repress the level of sulfite reductase activity in E. 2911 (26), Salmonella (27), Bacillus subtilis (26), Neurospora (28), and yeast (29). The evidence presented in this discussion strongly support the concept that sulfite reductase is an essential enzyme that catalyzes the biological reduction of sulfur. There is, however, some evidence that suggests that sulfite reductase may not be on the major pathway in all organisms. Schmidt and Schwenn (30) have reported the existence of an enzyme from Chlorella and spinach, which separates from 9 sulfite reductase during ammonium sulfate fractionation, that will reduce S-sulfoglutathione but not sulfite. S— sulfoglutathione contains the thiosulfuric acid group that has been proposed by Torii and Bandurski (21) to be the pro— duct of the PAPS reductase system. The formation of the S-sulfoglutathione from APS has been shown by Schiff and Hodson (10) and Schmidt (31) to be catalyzed by an enzyme they have called APS—sulfotransferase. The sulfotransferase catalyzes the same reaction as enzyme B of the PAPS reduc— tase system except that APS is the preferred substrate. The exact role of the thiosulfonate reductase in the reduction of sulfate has not been adequately determined, as the in 1139 substrate and the enzymatic product have yet to be characterized. It should be noted, however, that Schmidt 33 21' (32) have reported a Chlorella mutant, incapable of assimilating sulfate, which lacks thiosulfonate reductase activity, but has sulfite reductase activity. Properties of Sulfite Reductases from Various Organisms Cell free extracts, capable of catalyzing the six elec— tron reduction of sulfite to sulfide, have been obtained from E. 291; (33, 34), Salmonella tryphimurium (35), Saccharomyces cervisiae (23), spinach (36), Allium (37), A§l§ergillus nidulans (25), Neurospora crassa (24), Porphyra (38, 39), and dissimilatory sulfate reducing bacteria (40, 41) . The enzyme responsible for this reduction, sulfite reductase, has been purified from E. Coli (42), Aspergillus 10 (25),-Salmonella (43), saccharomyces (44), spinach (45), .and Allium (46). The physical, spectral, and catalytic pro— perties of the enzyme preparations are presented in Table 1. E. 221;, Salmonella, and yeast possess sulfite reductases which are similar in molecular weight, electron donors, reductions catalyzed, prosthetic groups and spectral pro- perties. There is some variation in the amount of flavins and iron present in these enzymes but their properties are essentially the same. The enzymes from Aspergillus, Allium and spinach also seem to constitute a separate group. They are lower molecular weight proteins which do not accept electrons from NADPH. They also lack the flavins and the capacity to catalyze the reduction of nitrite. It should be noted that the enzyme from Aspergillus may not belong with this group. Yashimoto 33 al. (25) have stated that the protein may have broken down during purifi— cation, and had lost the NADPH coupling site. Also the ability to use nitrite as a substrate was not examined due to experimental difficulties. From an evolutionary standpoint one would expect that the Aspergillus enzyme would be similar to the yeast enzyme, another fungus, as opposed to the enzymes from higher plantso The subunit structure of the enzymes from Salmonella and E. 99;; have been characterized by Siegel gt a}. (48) and Siegel and Davis (49) respectively. Both of these enzymes are composed of twelve polypeptide chains, eight "a" chains and four "8" chains. The polypeptides have a 11 .cossomscg no: mamaamsm mpmHQSOO .m .pSmmosu :aoposm wsapmcfiampgoo meow .e .chwssopoc p02 .0 .cosflasopmc mos +mmz Imoz .D .pscpsoo Sflbmae mm .m +mmz I mommz Ame” use 6mm an exam o- um I “mom >2 m 83.: asaaaa Ammmnsosnmsmv sa>nac oz +mmz.o mommz ooo.mw Amev mee.ouo.anewm.m cogs ms.o um 4.umom >2 Iooo.mm nomsaam Amwmusmmnasmv +mmz I mommz Ammw mmm.ono.auwm.m OI Q nm.II om >2 mm mN.: mafia“ mm mm was: m-m.a um oaanna anon s.ma sons :.Hm +wmmz + Imoz Asmmnemmv a2 ooo.:se «Hameosanm In maapna maIsa meosoaflm :Im sons HNION Mmmz I Imoz Asmmumwmuwsmv m2 ooo.oso Haoo .m Sosa mscssoz m +Nmmz ? Imoz Asmmnewmfimsmv a2 nooo.omm pmnms mofipammosa macaw Bonhamumo sosoc pgmfloz coszo Hmapommw OHPmSpmoam msoaposucm Sospooam amazoofloz m .msofipmhmgmsa owdpoScoa mpH%H5m emfiwfiasa czp mo mmfipscmosm .H manna 12 molecular weight between 50,000 to 60,000 with the "a" chains being a little heavier than the "B" chains. The en- zyme can be dissociated into two subunits with one subunit containing only "a" chains and the other subunit containing only "8" chains (49). The ” asubunit" can catalyze the re- duction of cytochrome c by NADPH while'the " Bsubunit" cata- lyzes the reduction of sulfite by reduced methyl viologen. The "a subunit" contains the flavins, while the " Bsubunit" contains the chromophore, iron, and acid labile sulfide. All of the sulfite reductases isolated have similar spectral properties (Table 1). The chromophore has been shown by several studies to be closely related to the active site of sulfite reduction (50, 51). The structure of the chromophore was determined by Murphy 23 al. (52) and shown to be a new type of heme, a siroheme. The only other enzyme that appears to have the same type of heme is nitrite re- ductase (53) which likewise catalyzes the six electron re- duction of an inorganic ion. The Mechanism of the Enzymatically Catalyzed Reduction of Sulfite The sequence of electron flow from the donor to the receptor molecules has been studied by Siegeland Kamin's group (48, 51, 54, 55) and Yashimoto and Sato (44, 50, 56). A proposed sequence for the electron flow through the enzyme molecule is shown below. 13 iron—sulfur . = gfiPup -9 s1roheme -9 SO3 MV MV NADPH -+ FAD —e FMN -9 The yeast, Eglgonella, and E. 99;} enzymes contain the whole sequence while the Aspergillug, Allium, and spinach enzymes lack the flavin containing subunit. The reduced methyl viologen could either donate electrons to the iron— sulfur component or to the siroheme or both. The siroheme passes the electrons to sulfite or other acceptors. The mechanism of the enzymatic reduction of sulfite to sulfide is very important in biology as there are three, or possibly four, enzymatically catalyzed six electron reduc— tions knwon. These enzymes include nitrogenase, nitrite reductase, sulfite reductase, and possibly thiosulfonate reductase. All biological nitrogen and sulfur have at one time been acted upon by these enzymes, thus the importance of six electron reductions is immense. Yet our knowledge about how the reductions are catalyzed is limited.at best. There has been considerable work on the mechanism of nitro— genase, but the basic instability of the enzyme and the lack of radioactive nitrogen compounds have prevented the eluci- dation of the reduction pathway. The studies on nitrite re- ductase have also had the same technical problems. Sulfite reductase, on the other hand, is easily isolated and purih fied. There is a readily available radioactive sulfur iso- tope and the enzyme is relatively stable. Thus the best EXPerimental system for studying enzyme—catalyzed, six 14 electron reduction should be sulfite reductase. Yet, only one published report by Kemp 2:.21 (34) has directly ad- dressed the mechanism of sulfite reduction. Their hypo- thesis was based on the belief that serine was involved at the catalytic site of sulfite reduction. It should be noted that many reaction mechanisms may be written for the reduction and the hypothesis presented by Kemp 23 2E (34) was without experimental support. Siegel and Kamin (51) and Siegel 33 a; (54) have published the only reports that give some potential insight into the mechanism of sulfite reduction. They reported that when radioactive sulfite was incubated with sulfite reductase and limiting amounts of NADPH, a tight enzyme— radioactive sulfur complex was formed. They concluded that the complex represented a covalent bond and hypothesized that this complex may represent a bound intermediate bet- ween sulfite and sulfide. The purpose of the studies presented in the next section was to gather information about the mechanism of sulfite reductase catalysis by attempting to isolate inter- mediates between sulfite and sulfide. These studies includ- ed binding experiments similar to the studies of Siegel and Kamin (51) described above and attempts to inactivate the enzyme such that only a partial reduction could be observed. MATERIALS AND METHODS Chemicals All of the chemicals (unless stated below) were reagent grade from commercial sources. Calcium phosphate gel was obtained from Bio-Rad Laboratories. The Sepharose 6B was purchased from Pharmacia Inc. The methyl viologen was from Mann Research Laboratories. The NaZBSSO3 was obtained from New England Nuclear Inc. Dr. G. Kennedy kindly provided the N,N,-dimethyl-p-phenylenediamine. Na S standard solu- 2 tions were prepared according to Asada (36). E. coli_Su1fite Reduggase PreparaEions The preparation of this enzyme is essentially that used by Siegel and Kamin (42) with modifications as outlined in the text. Cell Preparation E. 921; B (obtained from Dr. H. Sadoff of the Department of Microbiology at Michigan State University) was grown in the medium given by Siegel and Kamin (42) in two 6 1 batches, then transferred to a 100 l fermenter and grown to late log phase. The pH was maintained at 6.5 to 7.0 by periodic addi- tions of concentrated NHuOH. The cells were harvested in a Sharples centrifuge and the packed cells frozen and stored. 15 16 Extraction and Purification §§§p E. E. g2}; cells (30—100 g) were suspended in two volumes of 0.1 M K-phosphate buffer, pH 7.75, with 0.1 mM EDTA and disrupted in a Branson Sonifier (model 8—125) for 15 minutes in a 180 m1 Rosette cell cooled in a salt- ice bath. The suspension was centrifuged for 30 minutes at 15,000 x g, the supernatant was collected and this is re- ferred to as the crude extract. §§§p g. The protein content of the crude extract was determined and the pH of the extract adjusted to approx— imately 7.5 with 6 % (wt/vol) NaOH. Protamine sulfate (Sigma grade II), in a 5 % solution was adjusted to pH 7.0 using NHhOH, and was added to the crude extract so that the ratio of protamine sulfate to protein was 0.27. The precip- itate formed after 30 minutes of stirring was sedimented at 15,000 x g for 90 minutes and discarded. stgp,3. Ammonium sulfate (250 g) was slowly added to each liter of the supernatant solution. The mixture was stirred for 30 minutes and the pellet resulting from a 20 minute centrifugation at 6,000 x g was dissolved in sufficient 0.05 M K-phosphate buffer, pH 7.75. with 0.5 mM EDTA to yield a solution having a concentration of 20 mg/ml protein. Eggp 3. Calcium phosphate gel suspended in water (220 ml of a 30.3 mg dry weight of gel per ml solution) was added to each liter of protein solution and stirred for 30 minutes. The solution was then centrifuged for 20 minutes 17 at 6,000 x g and the pellet discarded. Twice as much gel solution as added previously was mixed with the supernatant and stirred for 30 minutes. The pellet resulting from a 20 minute centrifugation at 6,000 x g was then resuspended in 167 ml of 0.5 M K-phosphate buffer, pH 7.75, with 0.05 mM EDTA for each liter of original protein solution. The solution was stirred for 20 minutes and centrifuged for 20 minutes at 6,000 x g. The pellet was resuspended in 600 ml of 0.2 M K-phosphate buffer, pH 7.75, with 0.1 mM EDTA per liter of original protein solution and stirred for 30 minutes. The solution was then centrifuged at 6,000 x g for 20 minutes to sediment the gel. §£§p j. Ammonium sulfate (186 g) was added to each liter of gel eluate, stirred for 30 minutes and then centri- fuged for 20 minutes at 6,000 x g. An additional 45 g of ammonium sulfate was then added and the suspension stirred for 30 minutes. The pellet resulting from 20 minute centri— fugation at 6,000 x g was resuspended in a small volume of 0.1 M K-phosphate buffer, pH 7.75, with 0.1 mM EDTA and dialyzed for 16 hours against the same buffer. Eggp E. The dialyzed material was loaded on a 5 x 70 cm Sepharose 6B column equilibrated with 0.1 M K- phosphate buffer, pH 7.75, with 0.1 mM EDTA. The column was eluted with the same buffer at a flow rate of 10-15 ml/hour. E332 Z. The active fractions were pooled and precip- itated at 50 % ammonium sulfate with 30 minutes of stirring. 18 The pellet resulting from centrifugation at 6,000 x g for 20 minutes was resuspended in a minimal volume of 0.1 M K-phosphate buffer, pH 7.75, with 0.1 mM EDTA. FMN (1 mM), 170 pl, was then added and the solution dialyzed for 18 hours against 0.1 M K-phosphate buffer, pH 7.75, with 10 mM EDTA and then 18-24 hours with 0.1 mM K-phosphate buffer, pH 7.75, with 0.1 mM EDTA. Sulfite Reductase Assays Sulfiite-dependent NADPH Oxidase Assay The reaction mixture contained in umoles: K-phosphate buffer, pH 7.75, 100; NADPH, 0.2; NaZSOB, 0.5; and enzyme in a total volume of 1.0 ml. The total mixture minus the sulfite was incubated in a spectrophotometer and the endo- genous bleaching rate at 340 nm measured. Sulfite was tipped in and the rate of bleaching after tipping measured. The endogenous bleaching rate was subtracted from the rate with sulfite to obtain enzyme activity. One unit of enzyme was defined to be the amount of enzyme that would catalyze the oxidation of 1 umole NADPH per minute at 24-250C. This particular assay may not correctly reflect the level of sulfite reductase activity in crude extracts. As shown in the appendix, this activity may be associated With different reactions. _§jegel's Sulfide Production Assay This assay was a modification of the assay developed by Siegel (57). The reaction mixture contained in umoles: 19 K-phosphate buffer, pH 7.75, 100; glucose-6-P, 5; NADPH, 0.2; NaZSO 0.5; with 0.06 units glucose-6-P dehydrogenase and 39 enzyme in a total volume of 0.8 ml. The mixture was incubat- ed at 24-25OC for 30 minutes in one-half dram screw cap vials fitted with teflon liners. The reaction was stopped by adding 0.1 ml of 0.03 M FeC13 in 1.2 N HCl and 0.1 ml of 0.02 M N,N,-dimethyl-p-phenylenediamine in 7.2 N HCl. The cap was replaced immediately after the addition of the reagents. The optical density at 664 nm was measured after 30 minutes. NaZS was used to prepare a standard curve to relate OD664 to nmoles of H28 produced. Methyl Viologen Oxidation Assay The methyl viologen oxidation assay developed by Asada (36) was used in several preliminary studies and in the inhibition studies. The bottom of a glass Thunberg cuvette received 150 nmoles of K-phosphate buffer, pH 7.75, 2 mg BSA, and enzyme in a total volume of 0.95 ml. The side arm received 1.5 umole Na2803. The cuvette was evacu- ated and flushed with argon repeatedly until the cuvette was anaerobic. Reduced methyl viologen (0.6 ml of a 0.145 mg/ml solution) was transferred to the cuvette as pre- viously published (36, 58) and the endogenous bleaching rate at 604 nm was measured. The sulfite was then tipped in and the bleaching rate measured. Activity was defined to be the sulfite dependent bleaching rate. The reduced methyl viologen was made according to Asada (36). The number of m moles oxidized per unit time can be calculated by multi- plying the change in optical density by 1.143 x 10-“. 20 Protein Assays Eiuret Assay The method of Zamenhof (59) was used with the optical density of the colored product being read at 310 nm. BSA was used as a standard. Lowry Assay The protein was determined by the method of Lowry g; §l° (60), with BSA as a standard. Gel Electrophoresis Gel Prgparation and Running Procedures Davis gels. Electrophoresis gels (0.3 x 5 cm) were made and run using the solutions and methods of'Davis (61) except that the running buffer was one half as concentrated. After the gels were removed from the glass tubes they were stained according to the procedures described below. Erga gglg. The solutions and methods of Jovin 23 a1. (62) were used to make the gels. The enzyme preparation was treated with 45 ul of 0.05 M K-phosphate buffer, pH 7.75, 8 M urea, and 0.3 M mercaptoethanol for 12 hours at 24°C. A small sample of this mixture (20-40 pl) was mixed with 50 pl 4 % sucrose and loaded on the gels. The running buffers were as described by Jovin g: g}. (62). Bromophenol 'blue was used as a marker. EEainingyTechniques Coomassie Elue staining. The gel was placed in a test tube containing 50 % trichloroacetic acid for 1 hour. 21 The gel was then placed in 0.01 % Coomassie Blue in 50 % trichloroacetic acid for 1 hour and 20 minutes at 370C. Acetic acid (7 %) was used to destain the gels. Diaphorase stain. The gel was placed in a test tube containing 0.25 M K-phosphate buffer, pH 7.75, 2 mM NADPH, and 0.74 mg/ml nitrovblue tetrazolium until bands appeared. 3SS Binding Assay These procedures were those finally adopted to produce saturable binding. The reaction mixture which was in aluminum foil wrapped test tubes was started by adding the enzyme preparation and incubating for 5 minutes at 24°C. The reaction was stopped by placing the test tube in an ice bath. The samples were then loaded into the dialysis cells and the dialysis started. After 18 hours of dialysis the samples were removed and counted in Bray's scintillation fluid in a Beckman LS-133 Scintillation Counter using the 14C settings. A minus NADPH control was run in each experi- ment and this value was subtracted from the other values to obtain NADPH dependent binding. Dialysis Equipment and Running Procedures Two four-cavity 1 ml flow-through microdialysis cells (Bel-Art Products Model 347) were hooked in series so that the effluent of the first chamber of the first cell passed through the first chamber of the second cell before being discarded. The flow through the chambers was regulated by a peristaltic pump. The dialysis was performed with the 22 cells mounted horizontally on a shaking platform. A bubble left in the solution being dialyzed provided mixing during the operation. Unless otherwise stated in the figure or table legends the buffer used in the dialysis was 0.1 M K- phosphate, pH 7.75, with 0.1 mM EDTA. The flow rate for the first 4-6 hours was 40 ml per hour; the flow rate for the remaining 12-14 hours was 20 ml per hour. Each cavity of the cell was divided by one layer of dialysis membrane with Parafilm gaskets on both sides of the membrane. The membrane was prepared by the following treat- ments; the membrane was boiled for 15 minutes in 0.1 M K- phosphate buffer, pH 7.75, with 10 mM EDTA; left in 0.1 M K—phosphate buffer, pH 7.75, with 0.1 mM EDTA and 10 mM cysteine for 3 hours at 2500; treated with 0.1 M K-phosphate buffer, pH 7.75, with 0.1 mM EDTA and 10 mM Na2803 for 3.5 hours at 25°C; and finally the membrane was thoroughly wash- ed with 0.1 M K-phosphate buffer, pH 7.75, and 0.1 mM EDTA. Stoichiometry and Inhibition Assays The reaction mixture and procedures (except when modifi- ed as stated in the legend) described for the methyl viologen assay were used. The reaction was stopped by adding 0.15 ml of each of the Siegel reagents (described earlier) which had been placed in the neck of the Thunberg cuvette and Sealed with a cap. The vacuum in the cuvette pulled the reagents into the cuvette. After 30 minutes of shaking the reaction mixtures were removed and centrifuged for 5 minutes 23 to remove the precipitated protein and read at 664 nm in a spectrophotometer. A standard curve using Nags was prepared with the cuvettes which did not contain active protein. After reduced methyl viologen was added the tubes were made aerobic and Nags and the two Siegel reagents were added. The Nags could not be placed under a vacuum because of its volitility. The rest of the procedures described above were then followed. The ratio of sulfite-dependent methyl viologen oxidiz- ed and sulfiteadependent H28 produced Was then calculated. RESULTS Enzyme Purification The purification procedure of E. 39;; sulfite reductase was essentially the procedure developed by Siegel and Kamin (42) with the exception of the concentration step and the FMN activation step which were done at the end of the procedure. The FMN activation step was required to regain activity lost during the Sepharose 6B column and ammonium sulfate concentration step. A detailed description of the purification is presented in the methods section. A summary of a' typical purification is presented in Table 2. Certain data points are missing since it was not possible to assay all fractions due to volume constraints. The second ammonium sulfate fraction activity was too concentrated and the pro- tein in the Sepharose 6B column eluent was too dilute. Gel Electrophoresis of Purified Enzyme An attempt was made to establish the purity of the preparation using polyacrylamide disc gel electrophoresis. Seven bands were produced when the preparation was run on standard 7 % Ornstein—Davis gels and stained with Coomassie Blue (see Figure 2, number 1). These results were surpris- ing since the preparation was thought to be homogeneous. 24 25 .SOHpomm meompca esp Ga empaaomoc mam moascooosa one .pSmaHHcaNm mama mom coma mm: Aw omv mHHmo mace .w.vcxomm .mcma on p05 Bazoo wagoaosdmmoa mafiaob opmaSoo< .c .cosfisaopmc on p03 cameo .o .apfibfipom ommcaxo mmmfipom hpfi>fipom :Hoposg oSSHo> Soapomhm oaeaooam s Hesoe e aesoe III II 'III' -),!I'I III: .ommposcoa opH%H5m @o Sofipmofimflssm .m ofimme 26 awe/I ’5‘72')’ 54V: iii-5““ I _ ‘ IP- I I I Marking I _ Marking ___ j 7"" "' Dye "' " ' Dye '_—_7 1. Gel: Davis 2. Gel: Davis 3. Gel: Urea Stain: Coomassie Stain: Diaphorase Stain: Coomassie Blue Activity Blue Figure 2. Gel electrophoresis studies of sulfite reductase. The procedures used for these experiments are described in the methods section; 26.9 ug of sulfite reductase were used in each experiment. 27 An experiment was performed to find out which of the protein bands was sulfite reductase. Sulfite reductase has diaphorase activity and an assay for diaphorase is the enzyme dependent reduction of nitro-blue tetrazolium with NADPH. In the reduced form, nitro—blue tetrazolium is insoluble and thus makes a good gel stain. Figure 2 number 2 shows the banding pattern obtained with the activity stain. The five major bands stained with Coomassie Blue also stained with the diaphorase activity stain. E, 39;; sulfite reductase has twelve polypeptide chains of two molecular weights with a proposed ratio of 4 chains of one to 8 chains of the other polypeptide (49). A homo— geneous sulfite reductase preparation should show only 2 bands when denatured with urea, reduced with mercaptoethanol, and run on urea gels. Number 3 in Figure 2 shows that this was in fact the case. There was a minor third band that was just barely detectable. Its origin is unknown. The unusual siroheme present in the sulfite reductase (52) permits, by its spectral properties, the estimation of purity by the ratio of the amount of siroheme present to the amount of protein. The absorption at 278 nm reflects the amount of protein present while the optical densities at 386 and 587 nm represent the amount of the prosthetic group. Siegel and Kamin (42, 51) have reported that the 278:386z587 ratio for a homogeneous preparation is 3.6:1.0:0.23. The Sprotein preparation used in the electrophoresis experiments had_a.ratio of 3.62:1.0:0.22. 28 35 S Binding Studies Siegel and Kamin (51) reported in 1967 that in prelimi- nary experiments, radioactive sulfur from sulfite was bound to sulfite reductase when the reaction was run to completion with limiting amounts of NADPH. The following series of experiments were performed to verify and hopefully expand these studies. In this experimental design binding was defined as the amount of sulfur not removed from a reaction solution by extensive dialysis. Non-saturable Bindigg Initial experiments were hampered by high blank values which could not be significantly decreased by further dialy- sis. It was found that in these experiments the dialysis membranes 'became labeled. This high labeling, and, the high blank value could be reduced by pretreating the membranes with EDTA, cysteine and unlabeled Na2803' Table 3 shows the results from an experiment using the pretreated membranes. The binding was NADPH-dependent. The difference between the amount of binding, when limiting amounts of NADPH (Table 3, number 1) were used or excess amounts (Table 3, number 2) were used, showed that at least at the limiting concentration of NADPH no "satu— ration" of the binding had occurred. Figure 3a summarizes further experiments designed to study the saturation of this binding by holding the amount of sulfite used constant and in excess, while varying the amount of NADPH. Figure 3b .ma\mpflss mo.m mes QoHpmsmaoaa teensm esp mo hpabflpom oamfiooam one .QOHpoom weompos esp SH Hampoc SH confiscmcc can taseooosa psospMoApmsa escapees can SmpmAm mamaaeac one "sooammo some now ~50: \Hs om mo open Scam m Spas mazes ma non ccssflpsoo mamaamfie ems .maonamso mHmAHmHUOHOHs on» ma coomaa use comm pm mopssfis 0: Mom eomeSosH macs mmMSpNHs Soapomoa one 29 com teamso mamas m we 05mm a mum mmaaz mazes H mm teem m ome.ma mmm HmpOp n ma mmHos: H.o memm moaosa ooa Ams.s may states oposommsaIs moaosz ©.m Aoaos:\o: amvn ommm meos: mw:.o mmm Hdep m S .cofipomm meogpma mSp SH wonflsomow who mossumooag mfimhade USN msoflpfidsoo goapommh .ggmaw Qo Umpwpm mm mmmNZm 0m.:Om d P b I .P L) b )5 d 1 ‘ d "V00” ""00” WQ GS SeSG+GSSG SeOé + GSSG + H20 02+ 20H" 4GSH 3 lZGSH+1/202 2 GSSG+Se° "Jump" Kinetics time i 0T2 ' 034 ' 0T6 ' 033 p moles Sec;3 /ml Rxn.Mix. Optical density at 380 nm of Various con— centrations of sodium selenite in 10 mM reduced glutathione. The reaction mixture and conditions are des— cribed in the methods section. Figure 15. 94 The ATP sulfurylase-dependent increase in the optical density at 380 nm with time at various reduced glutathione concentrations. The reaction conditions were as described in the methods section. -—~———... . 95 Ow .. aoSEE we: ImOZEom a ImOZEo— . ImOEEm . ImOEEnN . .N.— .3 so.— Figure 16- 96 The effect of various reaction components on the production of elemental selenium (selenite equivalents) by ATP sulfurylase. The reaction conditions were as described in the methods section. The volume was 1.0 ml in all cases. 97 3.23va as E. On O? On ON O— )l“) L I O 0.53ch . ”Com- 6 ImOn . at? x D<Se\ 0 \0 Selenium trioxide Selenium trioxide is the anhydrous form of selenic acid and is readily converted to selenic acid when ex- posed to water. Schmidt and co—workers (141, 142) have reported that selenium trioxide reacts explosively with thiols forming thioselenic acids. The concept of a thiolytic cleavage of phosphoselenate anhydride thus seems very plausible if not probable. Alcoholysis of the selenium anhydride would also be predicted. Nissen and Benson (119) found ethyl selenate When plants fed selenate were extracted with hot ethanol. Ethyl selenate is the product expected of an alcoholytic cleavage of a~ selenium anhydride. As mentioned above, selenium trioxide is unstable in water. The enzymatic product should likewise be unstable. The enzymatic product is in fact unstable as shown by the great difficulty in detecting its forma- tion by either electrophoresis or column chromatography and the inability of glutathione to show any significant accumulation of this product (Figure 18). The thioselenic acid formed from the thiolytic cleavage could be easily converted to selenite (or 110 selenite equivalent) by the addition of one electron (one reduced glutathione). The conversion of organic thiosulfuric acids to sulfite and a disulfide by thiols is well documented. The evidence presented above can be summarized as follows: (1) Selenate could be converted in the presence of ATP, Mg++, ATP sulfurylase, reduced glutathione, and inorganic pyrophosphatase to elemental selenium through a compound that was functionally equivalent to selenite. (2) The stoichiometry of this conversion was the same as the stoichiometry observed for APS production from sulfate by ATP sulfurylase. Two moles of inorganic phosphate were released for each mole of product formed. (3) The properties associated with the enzymatic product were the same as the predicted properties of a phospho— selenate anhydride. These properties included the reactivity with thiol compounds and its basic instability in aqeous solutions. (H) It was possible, using known sulfur and selenium chemistry, to propose the reaction steps from a selenate anhydride to selenite (whose role as an intermediate in the overall reduction to elemental selenium was implied). (5) A compound can be detected which has the electrophoretic and acid lability properties of APS. This evidence, plus the evidence described in the literature showing the ability of selenate to be an effective substrate for ATP sulfurylase catalyzed reactions, led to the conclusion that APSe, or a compound 111 functionally equivalent to APSe was formed from selenate by ATP sulfurylase. The proposed mechanisms of the conversion from selenate to elemental selenium as described in these studies can be diagramed as shown in Figure 19. This proposal includes the thiolytic cleavage of a selenate anhydride, which due to the participation of ATP sulfury— lase (Figure 16) and the consumption of one ATP per one molecule of active selenium formed (Figure 17), was presumed to be APSe (either free or enzyme—bound). The conversion of the thioselenic acid to selenite is analo— gous to known sulfur chemistry. The mechanism of the catalytic oxidation of gluta— thione by selenite is the proposal made by Tsen and Tappel (138). The conversion of selenite to elemental selenium is essentially the mechanism proposed by Hsieh and Ganther (111). There is no direct evidence to dis— tinguish between two possible paths for the conversion of GS—SeH to elemental selenium. One possibility is presented in the diagram and the other possibility would be an intramolecular rearrangement forming elemental selenium and reduced glutathione (110). The rationale for including the production of hydrogen selenide in the proposed pathway was the fact that oxygen was required for elemental selenium formation. The proposed mechanism for the conversion of selenate to elemental selenium was designed to explain an in vitrg 112 9., 2m ‘0 - ‘8 SeO4‘ + ATP SU—AT’gY'ie. APSe+ PPi / GSH GSSeO§+AMP GSH GSSG arizo + _ GS+SG «fig—,L SeO§ GSSeSG cs‘r/ ‘2: 20H' case GSSG + Sec; + H20 GSSeH GSH 6556 + H25e 11/202 5e°+ H20 Figure 19. Proposed pathway for the ATP sulfurylase- catalyzed reduction of selenate. 113 phenomenon, but part of it has the potential of being a viable pathway for the reduction of selenate in XEXQ° The steps requiring oxygen would be side reactions of the reductive pathway. Although the reactions described can easily produce reduced selenium chemically, several of the reaction steps have been shown to be catalyzed by the enzyme glutathione reductase (110, 111). The catalyzed reactions couple the reducing power of NADPH to GS-Se-SG forming 2 GSH and HZSe. This catalysis reduces the amount of reduced glutathione required for the reaction. Several other enzymes which normally metabolize sulfur compounds may catalyze other reaction steps; i.g. the S—sulfoglutathione transhydrogenase described by Winell and Mannervick (193) could catalyze the conversion of GS—Se03' to 8e03=; APS sulfotransferase (10, 31) might catalyze the thiolytic cleavage, 222. If enzyme catalysis does occur in 1112 the mechanisms of the individual reaction steps would be different but the intermediates formed would be the same. This pathway would predict the observed antagonistic effects between selenate and sulfate as both would be substrate for ATP sulfurylase and presumably for the enzymes which synthesize and metabolize the sulfur amino acids. The inability to detect PAPSe or predicted PAPSe metabolite (selenate esters) would be explained as selenate would not form this intermediate. Also explained would be the inability of selenite to serve as an effective 114 substrate for sulfite reductase (the Km for sulfite and nitrite in E. £913 is 10LL times lower than the Km for selenite (34)). The observation that animals can reduce selenate, but not sulfate is likewise explained. In animals there is considerable evidence that the reduction of selenite is mediated by reduced glutathione (116) and the ability of ATP sulfurylase to form a product which can easily be converted to selenite would explain the ability of animals to utilize both selenite and selenate equally well (96). § The potential for such a pathway to be operable in biological organisms exists, but further in 3119 studies must be performed to decide whether this is the pathway that evolution has Selected. APPE NDIX APPENDIX SULFITE-DEPENDENT NADPH OXIDA§E ACTIVITY IN CULTURED TOBACCO CELL EXTRACTS INTRODUCTION ! This study presents data from a series of experiments on the sulfite—dependent NADPH oxidase activity present in crude extracts of cultured tobacco cells. Sulfite— dependent NADPH oxidase activity is often used to assay NADPH—linked sulfite reductase (42, 144), the enzyme which catalyzes the reduction of sulfite to hydrogen sulfide with the oxidation of three NADPH. The data presented in the following report show that sulfite reductase is not the only enzyme that can catalyze this oxidation. It was concluded that an enzyme system existed that was capable of initiating the self—perpetuating oxidation of sulfite. The sulfite oxidation chain reaction involved the inter— mediate production of superoxide radicals. NADPH, like many chemicals, can be oxidized by the radicals giving an enzyme—dependent and sulfite—dependent oxidation of NADPH. 115 MATERIALS AND METHODS We The materials used were laboratory reagent grade chemicals. Tobacco Extract Preparations Three to five day old tobacco cells were grown according to Filner (145), collected on a Buchner filter with Whatman No. 1 paper and rinsed with water. The cells were weighed and homogenized with fifteen strokes of a teflon plunger in a glass homogenizer. The homose- nizing medium was 1.5 x the weight of the cells and contained 0.1 M Tris-HCl, pH 7.5, with 1 mM cysteine. The homogenate was centrifuged for 20 minutes at 10,000 x g. The supernatant was made 45 % saturated with solid enzyme grade ammonium sulfate and the solution stirred for 30 minutes. The solution was centrifuged at 10,000 x g for 10 minutes. The supernatant solution, if boiled for 5 minutes is referred to as the boiled enzyme preparation. If the supernatant was dialyzed against 0.1 M Tris-HCl, pH 7.5, with 1 mM cysteine and 45 % saturation ammonium Sulfate for 14 hours, it is referred to as dialyzed enzyme Preparation. The boiled enzyme preparation used in these 116 117 experiments was derived from 5.2 g of cells, while the dia— ‘lyzed enzyme preparation was obtained from 4.9 g of cells. Sulfite—dependent NADPH oxidase Activity The complete reaction mixture contained, in nmoles: K—phosphate buffer, pH 7.4, 100; NADPH, 0.2; Na2803, 1.5; and varying amounts of boiled and dialyzed enzyme pre— parations in a total volume of 1.0 ml. The change in optical density at 340 nm was measured before the addition of sulfite and this value was subtracted from the rate . obtained after sulfite was added. Grant's Sulfite Assay The procedure used was that described by Bandurski (146) except that no protein precipitating agents were used. The 1.0 ml reaction mixture was added to 4 ml of color reagent and the optical density at 585 nm read after 10 minutes. The color reagent was freshly prepared before use by adding 2 ml of Fuchsin, 3 % (wt/vol) in 95 % ethanol, and 0.5 ml 40% formaldehyde to 120 ml of 0.8 M sulfuric acid. After 1 hour the mixture was filtered through Whatman No. 1 paper and used. Oxygen Uptake Studies These experiments were performed using a Clark—type Oxygen electrode (Yellow Springs Instrument Company) 118 covered with a Teflon membrane, that was kindly made available by Dr. Norman Good. The liquid capacity of the chamber was 2 ml. The signal generated was amplified and recorded. The mixture was kept at 190C for the duration of these experiments. RESULTS Preliminary studies (147) had indicated that cultured tobacco cell extracts contained a large amount of sulfite— dependent NADPH oxidase activity, an activity often used to monitor the enzyme NADPH sulfite reductase. This observation was confirmed and extended showing that the activity was heat labile and that dialysis removed a compound required for activity. Table 7 shows the acti- vation of the dialyzed preparation by a heat-treated pre— paration which presumably still contains the dialyzable cofactor, Table 7. The activation of dialyzed tobacco extract by boiled tobacco extract. Sulfite—dependent Preparation used NADPH oxidase activity AODBuO/minute 50 ul dialyzed prep. 0.0 50 ul boiled prep. 0.00338 50 ul boiled prep. + 50 ul dialyzed prep. 0.0358 The reaction conditions and enzyme preparations were as described in the methods section. The pH of the final mixture was 7.25. 119 120 The reaction had a rather precise pH optimum so care had to be taken that there was no change in the pH upon addition of other reactants. Figure 20 graphically pre- sents the pH optimum studies. The kinetic data obtained when the dialyzed pre— paration and sulfite concentrations were held constant and the concentration of the boiled preparation was varied, showed saturation. These data could be handled in the normal Michaelis—Menten sense. When the sulfite was varied and the other parameters held constant, there was no satura- tion and the shape of the curve showed that doubling the sulfite concentration would more than double the rate of NADPH oxidation. This last result was unexpected as all known sulfite reductases show normal Michaelis—Menten kinetics with respect to sulfite. The production of H28 is also required to define sulfite reductase activity. In this system there was no detectable production of H28 using Siegel's sulfide assay (57). The system itself did not prevent H28 from being assayed as a small amount of E. 22;; sulfite reductase added to the system produced the expected amount of H28. Thus the activity being measured was not sulfite reductase. The sulfite could be acting as a catalyst or could be consumed or metabolized during the reaction. This question was answered by the experiment presented in Table 8. The data show that sulfite disappearance is dependent 121 0.03- 0.02‘ E \ .C. ‘ E \ o <- m D 0.01- 0 <1 0'0 fir ' f I 6.0 6.5 7.0 7.5 8.0 8.5 p H Figure 20. pH optima for tobacco sulfite-dependent NADPH oxidase activity. The assay conditions were described in the methods section. The pH was measured at the conclusion of the assay. The enzyme prepara- tion used were 50 pl boiled extract from 5.2 g of cells and 50 pl dialyzed enzyme pre— paration from 4.9 g of cells. 122 Table 8. The disappearance of sulfite in the reaction mixtures. Treatment OD585 Complete system with 50 ul dialyzed enzyme . and 20 pl boiled enzyme 1.73 i 0.12 Complete system with 50 pl dialyzed enzyme , and 50 pl boiled enzyme ’0.97 i 0.03 50 H1 dialyzed enzyme and 50 pl boiled enzyme with NADPH and glucose—6—P being added after Grant's reagents 1.21 i 0.04 NADPH plus glucose-6—P with 50 ul dialyzed enzyme and 50 U1 boiled enzyme being added after Grant's reagents 2.97 i 0.05 Complete system with Na2803 being added after Grant's reagents 3.01 i 0.08 The conditions of the assay were the same as described in the methods section except that only 0.1 mole of Na2803 was used. The reaction was run for 30 minutes and was stopped by the addition of Grant's reagents. Glucose—6-P, NADPH, and Na SO were in a volume of 50 pl. Grant's sulfite assay is described in the methods section. 123 on the presence of the enzyme preparations. The presence or absence of reducing agent has no effect when the enzyme preparations are absent,and only a marginal effect when the enzymes are present. Table 9 demonstrates the con- sumption of the boiled enzyme preparation cofactor. The sulfite could be combining with a compound present in the boiled preparation or undergoing a reduction or oxidation reaction. The combination with an organic compound is unlikely as only a few such derivatives are known. There are three common inorganic compounds into which the sulfite could be converted; sulfide, elemental sulfur, and sulfate. Hydrogen sulfide was eliminated by previous studies and elemental sulfur was also eliminated as the reaction mixture did not become turbid during the reaction. The conversion of sulfite to sulfate is an oxidative pro- cess and a source of oxidizing potential could be molecular oxygen. This guess was substantiated by running the reaction anerobically. There was no change in the optical density at 340 nm until air was readmitted into the system. The oxygen dependency suggested the use of an oxygen electrode to study the consumption of oxygen during the reaction. The curves presented in Figure 21 show the oxygen consumption with various components of the complete reaction mixture. In all cases the oxygen con— sumption is sulfite-dependent and proceeds without the addition of reductant as does sulfite consumption. The 124 Table 9. Boiled enzyme factor consumption during the reaction. Treatment AODBQO/min/ml ___— _‘_—___.—‘—__..~__~— -_— , Complete reaction mixture with 50 ul dialyzed enzyme minus sulfite and boiled enzyme .4. + 1 pmole Na2803 5 U1 boiled enzyme -10 minutes and mixing 5 ul boiled enzyme 10 minutes and mixing 0.003 0.004 0.011 0.005 0.011 0.006 |+‘ l+ i i 0.0000 0.0009 0.0015 0.0008 0.0006 0.0006 the modification as stated in the table. The additions were made serially to the basic reaction mixture which was described in the methods section, with The activity was corrected for the dilution caused by the Volume of the additions. Figure 21. 125 0.1 pmole 02 EDTA 1-———c 1 minute Oxygen consumption with various reaction mixtures. Reaction A contained 1.8 ml of 0.1 M K—phosphate buffer, pH 7.4, 50 pl dialyzed enzyme, and 50 pl of boiled enzyme preparation. Reaction B con— tained 1.85 ml of 0.1 M K-phosphate buffer, pH 7.4, and 50 pl dialyzed enzyme. Reaction C con— tained 1.85 ml of 0.1 M K-phosphate buffer, pH 7.4, and 50 pl boiled enzyme. Reaction D con- tained only 1.9 ml of 0.1 M K-phosphate buffer, pH 7.4. Na 503 , 50 pl (2 pmoles) were added. EDTA, 50 pl (2 pmoles) were added. The methods section includes information about the oxygen electrode. 126 chelating reagent, EDTA, was added to these experiments because heavy metals, especially Mn++ are known to catalyze the oxidation of sulfite. Figure 21 shows that the mixture represented by curve A is the only system in which EDTA does not have an immediate effect. The mechanism of metal catalyzed sulfite oxidation is believed to include a superoxide radical. other experiments tested the effect of superoxide dismutase on the complete reac- tion and showed that it inhibited the oxygen consumption. Superoxide dismutase catalyzes the destruction of super- oxide radicals. Table 10 shows the effect of various additions on the sulfite—dependent NADPH oxidase activity. The data show that sulfite can be replaced by hydrogen peroxide at low concentrations but it becomes inhibitory at higher concentrations. The enzymes superoxide dismutase and catalase. also inhibited the reaction along with the reductant and free radical scavenger. ascorbic acid. 127 Table 10. Effect of various additions on NADPH oxidase activity. Enzyme Additions (serial) A 003pO/min/m1 a b 50 ul BE + 50 p1 DE none _ 0.006 - Na2803 + 0.5 nmoles SOj’ 0.053 + 10 Ml SODC 0.017 50 ul BE + 50 ul DE none 0.006 + 0.5 nmoles H282 0.044 + 1.0 nmoles H202 0.018 50 pl BE + 50 ul DE none g 0.004 - NaZSOB + 1.5 nmoles 803' 0.055 + 10 ul catalase 0.004 50 ul BE + 50 ul DE none 0.004 - Na2803 + 0.2 nmoles H202 0.052 + 0.2 nmoles H202 0.039 + 0.5 nmoles H202 0.024 50 pl BE + 50 ul DE none 0.038 - Na2803 + 1.5 nmoles 803: 0.045 + few grains ascorbate 0.002 a. Boiled enzyme. b. Dialyzed enzyme. 0. Super— oxide dismutase. See the legend of Table 9 for conditions. DISCUSSION Extracts of cultured tobacco cells have relatively large amounts of sulfite—dependent NADPH oxidase activity. The activity was heat labile, sulfite—dependent, and required a heat stable and dialyzable cofactor (see Table 7). Another characteristic of the reaction was its sharp pH optimum at 7.4. This activity is a common assay for NADPH sulfite reductase, however, several experiments eliminated the possibility that sulfite reductase Was responsible for the activity. The definitive result was that during the reaction no H23 was produced. It was possible, however, to show that sulfite was consumed during the reaction, along with the boiled enzyme cofactor (Table 8 and 9). Studies to determine the fate of the sulfite demon— strated that molecular oxygen was required for the reaction. The consumption of oxygen during the reaction was monitored. with an oxygen electrode. The oxidation of sulfite by oxygen is often a problem encountered when working with aqueous sulfite solutions. Heavy metals are effective catalysts for this oxidation and EDTA is routinely added to sulfite solutions to prevent this oxidation. The data presented in Figure 21 show that EDTA stops the consumption of oxygen in all the reaction 128 129 miXtures except the reaction mixture containing.both active enzyme (dialyzed enzyme) and cofactor (boiled enzyme). These results can be interpreted to mean that heavy metal—catalyzed sulfite oxidation is occurring in all cases except the complete reaction mixture which has a different mechanism for promoting the oxidation of sulfite. The oxidation of sulfite is a chain reaction (1.2. the oxidation of one sulfite promotes the oxidation of another sulfite, 232°) (148). The process has been res ported to involve the intermediate formation of a super- oxide radical (149). The remOVal of the superoxide radical would break the chain reaction and stop the oxi- dation process. Superoxide dismutase, which catalyzes the reaction 2 02" + 2 H+ -9 02 4 3202 (150) inhibits both the oxygen uptake in the complete reaction mixture and the sulfite-dependent NADPH oxidation (Table 10). The free radical scavenger, ascorbate (151), likewise inhibits the NADPH oxidase activity. Hydrogen peroxide (as shown in Table 10) can replace sulfite in oxidizing NADPH when used at low concentrations, but at higher concentrations becomes inhibitory. Catalase also in— hibits the sulfite-dependent NADPH oxidation, indicating a requirement for H202. The ability of the free radicals involved in sulfite oxidation to oxidize reduced NADPH to NADP+ was shown by Klebanoff (152) in 1961. There are presently several 130 enzyme systems known which in the presence of their sub— strate initiate sulfite oxidation (150, 153). The sys— tems include xanthine oxidase, aldehyde oxidase, cyto- chrome oxidase, lipoxygenase and peroxidase. While the role of H202 in the xanthine oxidase system can be eliminated, it has been shown that the peroxidase system absolutely requires the presence of H202 (152, 153). 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