Identification and reactivity of the catalytic site of pig liver thioltransferase

The active site cysteine of pig liver thioltransferase was identified as Cys22. The kinetics of the reaction between Cys22 of the reduced enzyme and iodoacetic acid as a function of pH revealed that the active site sulfhydryl group had a pKa of 2.5. Incubation of reduced enzyme with [1-14C]cysteine prevented the inactivation of the enzyme by iodoacetic acid at pH 6.5, and no stable protein-cysteine disulfide was found when the enzyme was separated from excess [1-14C]cysteine, suggesting an intramolecular disulfide formation. The results suggested a reaction mechanism for thioltransferase. The thiolated Cys22 first initiates a nucleophilic attack on a disulfide substrate, resulting in the formation of an unstable mixed disulfide between Cys22 and the substrate. Subsequently, the sulfhydryl group at Cys25 is deprotonated as a result of micro-environmental changes within the active site domain, releasing the mixed disulfide and forming an intramolecular disulfide bond. Reduced glutathione, the second substrate, reduces the intramolecular disulfide forming a transient mixed disulfide which is then further reduced by glutathione to regenerate the reduced enzyme and form oxidized glutathione. The rate-limiting step for a typical reaction between a disulfide and reduced glutathione is proposed to be the reduction of the intramolecular disulfide form of the enzyme by reduced glutathione.     

Significance of redox-active cysteines in human FAD synthase isoform 2

FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is the last enzyme in the pathway converting riboflavin into FAD. In humans, FADS is localized in different subcellular compartments and exists in different isoforms. Isoform 2 (490-amino acids) is organized in two domains: the 3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and one resembling a molybdopterin-binding (MPTb) domain, with a hypothetical regulatory role. hFADS2 contains ten Cys residues, seven of which located in the PAPS reductase domain, with a possible involvement either in FAD synthesis or in FAD delivery to cognate apo-flavoproteins. A homology model of the PAPS reductase domain of hFADS2 revealed a co-ordinated network among the Cys residues in this domain. In this model, C312 and C303 are very close to the flavin substrate, consistent with a significantly lowered FAD synthesis rate in C303A and C312A mutants. FAD synthesis is also inhibited by thiol-blocking reagents, suggesting the involvement of free cysteines in the hFADS2 catalytic cycle. Mass spectrometry measurements and titration with thiol reagents on wt hFADS2 and on several individual cysteine/alanine mutants allowed us to detect two stably reduced cysteines (C139 and C241, one for each protein domain), two stable disulfide bridges (C399–C402, C303–C312, both in the PAPS domain), and two unstable disulfides (C39–C50; C440–C464). Whereas the C39–C50 unstable disulfide is located in the MPTb domain and appears to have no catalytic relevance, a cysteine-based redox switch may involve formation and breakdown of a disulfide between C440 and C464 in the PAPS domain.     

Thiol/disulfide redox equilibrium and kinetic behavior of chicken liver fatty acid synthase

Chicken liver fatty acid synthase is rapidly inactivated and cross-linked at pH 7.2 and 8.0 by incubation with low concentrations of common biological disulfides including glutathione disulfide, coenzyme A disulfide, and glutathione-coenzyme A-mixed disulfide. Glutathione disulfide inactivation of the enzyme is accompanied by the oxidation of a total of 4-5 enzyme thiols per monomer. Only one glutathione equivalent is incorporated per monomer as a protein-mixed disulfide, and its rate of incorporation is significantly slower than the rate of inactivation. The formation of protein-SS-protein disulfides results in significant cross-linking of enzyme subunits. The inactive enzyme is rapidly and completely reactivated, and the cross-linking is completely reversed by incubation of the enzyme with thiols (10-20 mM) including dithiothreitol, mercaptoethanol, and glutathione. In a glutathione redox buffer (GSH + GSSG), disulfide bond formation comes to equilibrium. The enzyme activity at equilibrium is dependent both on the ratio of glutathione to glutathione disulfide and on the total glutathione concentration. The equilibrium constant for the redox equilibration of fatty acid synthase in a glutathione redox buffer is 15 mM (Ered + GSSG in equilibrium Eox + 2GSH). The formation of at least one protein-protein disulfide per monomer dominates the redox properties of the enzyme while the formation of one protein-mixed disulfide with glutathione (Kmixed = 0.45) has little effect on activity. The oxidation equilibrium constant suggests that there would be no significant cycling between the reduced and the oxidized enzyme in response to likely physiological variations in the hepatic glutathione status. The possibility that changes in the concentration of cellular glutathione may act as a mechanism for metabolic control of other enzymes is discussed.     

Redox, mutagenic and structural studies of the glutaredoxin/arsenate reductase couple from the cyanobacterium Synechocystis sp. PCC 6803

The arsenate reductase from the cyanobacterium Synechocystis sp. PCC 6803 has been characterized in terms of the redox properties of its cysteine residues and their role in the reaction catalyzed by the enzyme. Of the five cysteines present in the enzyme, two (Cys13 and Cys35) have been shown not to be required for catalysis, while Cys8, Cys80 and Cys82 have been shown to be essential. The as-isolated enzyme contains a single disulfide, formed between Cys80 and Cys82, with an oxidation-reduction midpoint potential (E(m)) value of -165mV at pH 7.0. It has been shown that Cys15 is the only one of the four cysteines present in Synechocystis sp. PCC 6803 glutaredoxin A required for its ability to serve as an electron donor to arsenate reductase, while the other three cysteines (Cys18, Cys36 and Cys70) play no role. Glutaredoxin A has been shown to contain a single redox-active disulfide/dithiol couple, with a two-electron, E(m) value of -220mV at pH 7.0. One cysteine in this disulfide/dithiol couple has been shown to undergo glutathionylation. An X-ray crystal structure, at 1.8? resolution, has been obtained for glutaredoxin A. The probable orientations of arsenate reductase disulfide bonds present in the resting enzyme and in a likely reaction intermediate of the enzyme have been examined by in silico modeling, as has the surface environment of arsenate reductase in the vicinity of Cys8, the likely site for the initial reaction between arsenate and the enzyme.     

Properties and utility of the peculiar mixed disulfide in the bacterial glutathione transferase B1-1

Bacterial glutathione transferases appear to represent an evolutionary link between the thiol:disulfide oxidoreductase and glutathione transferase superfamilies. In particular, the observation of a mixed disulfide in the active site of Proteus mirabilis glutathione transferase B1-1 is a feature that links the two families. This peculiar mixed disulfide between Cys10 and one GSH molecule has been studied by means of ESR spectroscopy, stopped-flow kinetic analysis, radiochemistry, and site-directed mutagenesis. This disulfide can be reduced by dithiothreitol but even a thousand molar excess of GSH is poorly effective due to an unfavorable equilibrium constant of the redox reaction (K(eq) = 2 x 10(-4)). Although Cys10 is partially buried in the crystal structure, in solution it reacts with several thiol reagents at a higher or comparable rate than that shown by the free cysteine. Kinetics of the reaction of Cys10 with 4,4'-dithiodipyridine at variable pH values is consistent with a pK(a) of 8.0 +/- 0.1 for this residue, a value about 1 unit lower than that of the free cysteine. The 4,4'-dithiodipyridine-modified enzyme reacts with GSH in a two-step mechanism involving a fast precomplex formation, followed by a slower chemical step. The natural Cys10-GSH mixed disulfide exchanges rapidly with free [3H]GSH in a futile redox cycle in which the bound GSH is continuously replaced by the external GSH. Our data suggest that the active site of the bacterial enzyme has intermediate properties between those of the recently evolved glutathione transferases and those of the thiol:disulfide oxidoreductase superfamily.     

Crystal structure of the DsbB-DsbA complex reveals a mechanism of disulfide bond generation

Oxidation of cysteine pairs to disulfide requires cellular factors present in the bacterial periplasmic space. DsbB is an E. coli membrane protein that oxidizes DsbA, a periplasmic dithiol oxidase. To gain insight into disulfide bond formation, we determined the crystal structure of the DsbB-DsbA complex at 3.7 A resolution. The structure of DsbB revealed four transmembrane helices and one short horizontal helix juxtaposed with Cys130 in the mobile periplasmic loop. Whereas DsbB in the resting state contains a Cys104-Cys130 disulfide, Cys104 in the binary complex is engaged in the intermolecular disulfide bond and captured by the hydrophobic groove of DsbA, resulting in separation from Cys130. This cysteine relocation prevents the backward resolution of the complex and allows Cys130 to approach and activate the disulfide-generating reaction center composed of Cys41, Cys44, Arg48, and ubiquinone. We propose that DsbB is converted by its specific substrate, DsbA, to a superoxidizing enzyme, capable of oxidizing this extremely oxidizing oxidase.     

Mechanism of the electron transfer catalyst DsbB from Escherichia coli

The membrane protein DsbB from Escherichia coli is essential for disulfide bond formation and catalyses the oxidation of the periplasmic dithiol oxidase DsbA by ubiquinone. DsbB contains two catalytic disulfide bonds, Cys41-Cys44 and Cys104-Cys130. We show that DsbB directly oxidizes one molar equivalent of DsbA in the absence of ubiquinone via disulfide exchange with the 104-130 disulfide bond, with a rate constant of 2.7 x 10 M(-1) x s(-1). This reaction occurs although the 104-130 disulfide is less oxidizing than the catalytic disulfide bond of DsbA (E(o)' = -186 and -122 mV, respectively). This is because the 41-44 disulfide, which is only accessible to ubiquinone but not to DsbA, is the most oxidizing disulfide bond in a protein described so far, with a redox potential of -69 mV. Rapid intramolecular disulfide exchange in partially reduced DsbB converts the enzyme into a state in which Cys41 and Cys44 are reduced and thus accessible for reoxidation by ubiquinone. This demonstrates that the high catalytic efficiency of DsbB results from the extreme intrinsic oxidative force of the enzyme.     

Oxidation-reduction properties of two engineered redox-sensitive mutant Escherichia coli malate dehydrogenases

Redox potentials for two inactivating intrasubunit disulfides that link helix-5 and helix-9 in mutant Escherichia coli malate dehydrogenases have been determined. The Em is -285 mV when cysteines are at positions 121 and 305 and -295 mV when the cysteines are at positions 122 and 305. Oxidation to the disulfide affects kcat but not Km values. In the single V121C and N122C mutants, the Cys in helix-5 affects the Km for oxalacetate. The pH optimum in the direction of malate formation is affected by the redox state of the enzyme. Clearly, a disulfide bond can and does form between Cys residues substituted into positions 121 or 122 in the nucleotide binding domain and 305 in the carbon substrate binding domain of this NAD-dependent malate dehydrogenase. Apparently, crosslinking the domains interferes with catalysis.     

Cell polarization during monopolar cytokinesis

The biogenesis of mitochondrial intermembrane space proteins depends on specific machinery that transfers disulfide bonds to precursor proteins. The machinery shares features with protein relays for disulfide bond formation in the bacterial periplasm and endoplasmic reticulum. A disulfide-generating enzyme/sulfhydryl oxidase oxidizes a disulfide carrier protein, which in turn transfers a disulfide to the substrate protein. Current views suggest that the disulfide carrier alternates between binding to the oxidase and the substrate. We have analyzed the cooperation of the disulfide relay components during import of precursors into mitochondria and identified a ternary complex of all three components. The ternary complex represents a transient and intermediate step in the oxidation of intermembrane space precursors, where the oxidase Erv1 promotes disulfide transfer to the precursor while both oxidase and precursor are associated with the disulfide carrier Mia40.     

Continuous fluorometric assay of phenol sulfotransferase.

Phenol sulfotransferases (EC 2.8.2.1) catalyze the sulfation of the acceptor hydroxyl group using 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the donor substrate. Previous assays of these enzymes, which exhibit varied acceptor substrate specificities, have required termination of the catalysis followed by isolation and quantitation of formed sulfate ester. In this report, the sulfation of the fluorescent compound, resorufin, is investigated. Reaction of PAPS with resorufin, catalyzed by bovine lung phenol sulfotransferase, bleaches the emission of this acceptor at the pH of the reaction (pH 6.4 optimum). It is thereby possible to continuously record the sulfation reaction. Analysis of single progress curves by integrated replot can be used to determine the initial velocities and also indicates the formation of a product inhibitor, probably resorufin sulfate ester, with Ki less than Km. Sensitivity of the reaction is less than 1 pmol/min. The maximal rate of resorufin sulfation by the bovine lung enzyme is estimated at 57 nmol/mg/min, which is 10% of the rate with an optimal substrate 2-naphthol. This assay may be most sensitive for phenol sulfotransferases with optimal activities at greater than pH 6, due to the acid-base properties of resorufin (pK alpha 6), which becomes nonfluorescent upon protonation.     

Redox, mutagenic and structural studies of the glutaredoxin/arsenate reductase couple from the cyanobacterium Synechocystis sp. PCC 6803

The arsenate reductase from the cyanobacterium Synechocystis sp. PCC 6803 has been characterized in terms of the redox properties of its cysteine residues and their role in the reaction catalyzed by the enzyme. Of the five cysteines present in the enzyme, two (Cys13 and Cys35) have been shown not to be required for catalysis, while Cys8, Cys80 and Cys82 have been shown to be essential. The as-isolated enzyme contains a single disulfide, formed between Cys80 and Cys82, with an oxidation-reduction midpoint potential (E_m) value of − 165 mV at pH 7.0. It has been shown that Cys15 is the only one of the four cysteines present in Synechocystis sp. PCC 6803 glutaredoxin A required for its ability to serve as an electron donor to arsenate reductase, while the other three cysteines (Cys18, Cys36 and Cys70) play no role. Glutaredoxin A has been shown to contain a single redox-active disulfide/dithiol couple, with a two-electron, E_m value of − 220 mV at pH 7.0. One cysteine in this disulfide/dithiol couple has been shown to undergo glutathionylation. An X-ray crystal structure, at 1.8 Å resolution, has been obtained for glutaredoxin A. The probable orientations of arsenate reductase disulfide bonds present in the resting enzyme and in a likely reaction intermediate of the enzyme have been examined by in silico modeling, as has the surface environment of arsenate reductase in the vicinity of Cys8, the likely site for the initial reaction between arsenate and the enzyme.     

Kinetic studies on a 15-hydroxyprostaglandin dehydrogenase from human placenta.

Kinetic studies have shown that the reaction catalyzed by the human placental 15-hydroxyprostaglandin dehydrogenase proceeds by a single displacement mechanism. Addition of the reactants is ordered with NAD⁺ binding first. The lifetime of the ternary complex is affected by the pH of the reaction mixture. At pH 7.0 a kinetically significant ternary complex is formed, while at pH 9.0 the ternary complex is not kinetically significant (Theorell-Chance mechanism). There is evidence for the occurrence of a kinetically significant isomerization of the enzyme · NADH complex at pH 9.0 but not at pH 7.0. At high substrate concentrations there is formation of unreactive complexes between the 15-hydroxyrostaglandin and both the free enzyme and enzyme · NADH complex and between the 15-ketoprostaglandin and both the free enzyme and enzyme · NAD⁺ complex. The inhibition of the 15-hydroxyprostaglandin dehydrogenase by various prostaglandins and prostaglandin analogs may be explained by the formation of similar unreactive complexes. Certain prostaglandin analogs, arachidonic acid, and ethacrynic acid also affect the activity of the enzyme by causing its irreversible inactivation.     

Purification and kinetic characterization of a phenol-sulfating form of phenol sulfotransferase from human brain

The identification of three forms of phenol sulfotransferase (PST) in human brain and the subsequent purification and kinetic characterization of a phenol-sulfating form of the enzyme are described. Two forms of PST which were capable of conjugating phenol and a third form which sulfated dopamine were resolved from one another using DEAE-cellulose chromatography. One of the phenol-sulfating forms (P1-PST) was subsequently purified on Affi-Gel blue and Sephacryl S-200, giving a final purification of almost 390-fold, with an overall yield of approximately 5%. The purified enzyme was sensitive to NaCl and showed an optimum for phenol conjugation at pH 8.5. Kinetic analysis demonstrated that sulfation by P1-PST proceeds via a sequential ordered, bi-substrate reaction mechanism, where 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is the leading substrate. The true Km and Kia values for PAPS were both 0.35 microM, while the true Km value for phenol was 2.8 microM.     

Regulation of the catalytic activity and structure of human thioredoxin 1 via oxidation and S-nitrosylation of cysteine residues

The mammalian cytosolic/nuclear thioredoxin system, comprising thioredoxin (Trx), selenoenzyme thioredoxin reductase (TrxR), and NADPH, is the major protein-disulfide reductase of the cell and has numerous functions. The active site of reduced Trx comprises Cys(32)-Gly-Pro-Cys(35) thiols that catalyze target disulfide reduction, generating a disulfide. Human Trx1 has also three structural Cys residues in positions 62, 69, and 73 that upon diamide oxidation induce a second Cys(62)-Cys(69) disulfide as well as dimers and multimers. We have discovered that after incubation with H(2)O(2) only monomeric two-disulfide molecules are generated, and they are inactive but able to regain full activity in an autocatalytic process in the presence of NADPH and TrxR. There are conflicting results regarding the effects of S-nitrosylation on Trx antioxidant functions and which residues are involved. We found that S-nitrosoglutathione-mediated S-nitrosylation at physiological pH is critically dependent on the redox state of Trx. Starting from fully reduced human Trx, both Cys(69) and Cys(73) were nitrosylated, and the active site formed a disulfide; the nitrosylated Trx was not a substrate for TrxR but regained activity after a lag phase consistent with autoactivation. Treatment of a two-disulfide form of Trx1 with S-nitrosoglutathione resulted in nitrosylation of Cys(73), which can act as a trans-nitrosylating agent as observed by others to control caspase 3 activity (Mitchell, D. A., and Marletta, M. A. (2005) Nat. Chem. Biol. 1, 154-158). The reversible inhibition of human Trx1 activity by H(2)O(2) and NO donors is suggested to act in cell signaling via temporal control of reduction for the transmission of oxidative and/or nitrosative signals in thiol redox control.     

The Role of Cysteine Residues in Redox Regulation and Protein Stability of Arabidopsis thaliana Starch Synthase 1

Starch biosynthesis in Arabidopsis thaliana is strictly regulated. In leaf extracts, starch synthase 1 (AtSS1) responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1) that specific thiol-disulfide exchange in AtSS1 influences its catalytic function 2) that each conserved Cys residue has an impact on AtSS1 catalysis. Recombinant AtSS1 versions carrying combinations of cysteine-to-serine substitutions were generated and characterized in vitro. The results demonstrate that AtSS1 is activated and deactivated by the physiological redox transmitters thioredoxin f1 (Trxf1), thioredoxin m4 (Trxm4) and the bifunctional NADPH-dependent thioredoxin reductase C (NTRC). AtSS1 displayed an activity change within the physiologically relevant redox range, with a midpoint potential equal to -306 mV, suggesting that AtSS1 is in the reduced and active form during the day with active photosynthesis. Cys164 and Cys545 were the key cysteine residues involved in regulatory disulfide formation upon oxidation. A C164S_C545S double mutant had considerably decreased redox sensitivity as compared to wild type AtSS1 (30% vs 77%). Michaelis-Menten kinetics and molecular modeling suggest that both cysteines play important roles in enzyme catalysis, namely, Cys545 is involved in ADP-glucose binding and Cys164 is involved in acceptor binding. All the other single mutants had essentially complete redox sensitivity (98–99%). In addition of being part of a redox directed activity “light switch”, reactivation tests and low heterologous expression levels indicate that specific cysteine residues might play additional roles. Specifically, Cys265 in combination with Cys164 can be involved in proper protein folding or/and stabilization of translated protein prior to its transport into the plastid. Cys442 can play an important role in enzyme stability upon oxidation. The physiological and phylogenetic relevance of these findings is discussed.     

Efficient approach to synthesis of two-chain asymmetric cysteine analogs of receptor-binding region of transforming growth factor-alpha.

The putative receptor-binding region of human transforming growth factor-alpha (TGF alpha) has been shown to be contributed by two fragments: an A-chain (residue 12-18) and a 17-residue carboxyl fragment (residue 34-50) that includes a disulfide-containing C-loop (residue 34-43). An approach to the synthesis of two-chain analogs containing an intermolecular disulfide linked A-chain and the 17-residue carboxyl fragment (C-fragment) possessing receptor-binding activity is described. The synthesis was achieved by the solid-phase method using the Boc-benzyl protecting group strategy. The single Cys of the A-chain was activated as a mixed disulfide with 2-thiopyridine to form the intermolecular disulfide bond with Cys41 or Cys46 of the C-fragment on the resin support. Prior to this reaction, the acetamido (Acm) protecting group of Cys41 or Cys46 was removed by Hg(OAc)2 on the resin support. The peptide and side chain protecting groups including the S-methylbenzyl moiety of the Cys34 and Cys43 were concomitantly cleaved by high HF. The intramolecular disulfide with two unprotected Cys was formed in the presence of an intermolecular disulfide. This intramolecular disulfide bond formation was usually not feasible under the traditionally-held scheme at basic pH since disulfide interchange would occur faster than intramolecular oxidation. To prevent the disulfide interchange, a new method was devised. The intramolecular disulfide bond oxidation was mediated by dimethylsulfoxide at an acidic pH, at which the disulfide interchange reaction was suppressed. The desired product was obtained with a 60-70% yield.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic studies on pig heart cytoplasmic malate dehydrogenase.

Kinetic studies on the pig heart cytoplasmic malate dehydrogenase have been performed over a wide range of conditions using the full time course of the reaction and computer simulation to obtain the kinetic parameters. The maximum velocity and Michaelis constants for the oxidation of reduced coenzyme have been determined as a fundtion of pH in 0.05 M phosphate buffer at 15 degrees. At pH 7.5 and at low substrate concentrations, the kinetic data are consistent with a sequential addition of substrates, coenzyme binding first, and involving the formation of at least one ternary complex. No oxalacetate binding to the enzyme was observed. The rate constants for the dissociation of coenzyme from the enzyme-coenzyme complex are small enough to define the maximum velocity in either direction of the reaction. These data, plus data using deuterated reduced coenzyme, indicate that the chemical transformation step is not rate determining. It is also shown that DPNH binding can be tight enough to practically exclude the possibility of obtaining initial velocities when measuring the reduction of DPN. Kinetic abnormalities do appear at higher substrate or product concentrations, but these do not appear to be related to the formation of inactive abortice, complexes.     

Substrate inhibition in human hydroxysteroid sulfotransferase SULT2A1: studies on the formation of catalytically non-productive enzyme complexes

The cytosolic sulfotransferase hSULT2A1 is the major hydroxysteroid (alcohol) sulfotransferase in human liver, and it catalyzes the 3′-phosphoadenosine-5′-phosphosulfate (PAPS)-dependent sulfation of various endogenous hydroxysteroids as well as many xenobiotics that contain alcohol and phenol functional groups. The hSULT2A1 often displays substrate inhibition, and we have hypothesized that a key element in this response to increasing substrate concentration is the formation of non-productive ternary dead-end enzyme complexes involving the nucleotide product, adenosine 3′,5′-diphosphate (PAP). One of these substrates for hSULT2A1 is dehydroepiandrosterone (DHEA), a major circulating steroid hormone in humans that serves as precursor to both androgens and estrogens. We have utilized DHEA in both initial velocity studies and equilibrium binding experiments in order to evaluate the potential role of ternary complexes in substrate inhibition of the enzyme. Our results indicate that hSULT2A1 forms non-productive ternary complexes that involve either DHEA or dehydroepiandrosterone sulfate, and the formation of these ternary complexes displays negative cooperativity in the binding of DHEA.     

Egg white sulfhydryl oxidase: kinetic mechanism of the catalysis of disulfide bond formation

The flavin-dependent sulfhydryl oxidase from chicken egg white catalyzes the oxidation of sulfhydryl groups to disulfides with reduction of oxygen to hydrogen peroxide. The oxidase contains FAD and a redox-active cystine bridge and accepts a total of 4 electrons per active site. Dithiothreitol (DTT; the best low molecular weight substrate known) reduces the enzyme disulfide bridge with a limiting rate of 502/s at 4 degrees C, pH 7.5, yielding a thiolate-to-flavin charge-transfer complex. Further reduction to EH4 is limited by the slow internal transfer of reducing equivalents from enzyme dithiol to oxidized flavin (3.3/s). In the oxidative half of catalysis, oxygen rapidly converts EH4 to EH2, but Eox appearance is limited by the slow internal redox equilibration. During overall turnover with DTT, the thiolate-to-flavin charge-transfer complex accumulates with an apparent extinction coefficient of 4.9 mM-1 cm-1 at 560 nm. In contrast, glutathione (GSH) is a much slower reductant of the oxidase to the EH2 level and shows a kcat/Km 100-fold smaller than DTT. Full reduction of EH2 by GSH shows a limiting rate of 3.6/s at 4 degrees C comparable to that seen with DTT. Reduced RNase is an excellent substrate of the enzyme, with kcat/Km per thiol some 1000- and 10-fold better than GSH and DTT, respectively. Enzyme-monitored steady-state turnover shows that RNase is a facile reductant of the oxidase to the EH2 state. This work demonstrates the basic similarity in the mechanism of turnover between all of these three substrates. A physiological role for sulfhydryl oxidase in the formation of disulfide bonds in secreted proteins is discussed.