Biotin sulfoxide reductase: Tryptophan 90 is required for efficient substrate utilization

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase (BSOR) catalyzes the reduction of d-biotin d-sulfoxide to biotin and contains the molybdopterin guanine dinucleotide (MGD) cofactor as its sole prosthetic group. Comparison of the primary sequences of BSOR and the closely related enzyme dimethyl sulfoxide reductase (DMSOR) indicated a number of conserved residues, including an active-site tryptophan residue (W90), which has been suggested to be involved in hydrogen bonding to the oxo group on the Mo(VI) center in BSOR. Site-directed mutagenesis has been used to replace tryptophan 90 in BSOR with phenylalanine, tyrosine, and alanine residues to examine the role of this residue in catalysis. All three BSOR mutant proteins were purified to homogeneity and contained MGD. The mutant proteins retained very limited activity toward the oxidizing substrates tested, with W90F retaining the most activity (3.4% of wild type). All three W90 mutant proteins exhibited greatly reduced k(cat) values compared to that of the wild-type enzyme, which was accompanied by little change in K(mapp). In addition, the mutant proteins had perturbed visible absorption and circular dichroism spectra suggesting different oxidation states of the Mo center. Purified samples of wild-type BSOR did not exhibit electron paramagnetic resonance (EPR) signals indicating a Mo(VI) center. After redox-cycling, partially reduced samples of wild-type BSOR revealed a proton-split S=1/2 Mo(V) resonance (g(1,2,3)=1.999, 1.981, 1.967; A(1,2,3)=1.40, 1.00, 1.05 mT) analogous to that observed in DMSOR. In contrast, EPR studies of the purified W90 mutant proteins revealed distinct S=1/2 Mo(V) resonances that were resistant to both oxidation and reduction, indicating that the Mo was trapped in the intermediate Mo(V) oxidation state. These results strongly suggest that W90 in BSOR plays a critical role in catalysis by serving as a hydrogen bond donor to the oxo group on the Mo(VI) center.     

Studies on the interaction of NADPH with Rhodobacter sphaeroides biotin sulfoxide reductase

Rhodobacter sphaeroides biotin sulfoxide reductase (BSOR) contains the bis(molybdopterin guanine dinucleotide)molybdenum cofactor and catalyzes the reduction of D-biotin-D-sulfoxide to biotin. This protein is the only member of the dimethyl sulfoxide reductase family of molybdopterin enzymes that utilizes NADPH as the direct electron donor to the catalytic Mo center. Kinetic studies using stopped-flow spectrophotometry indicate that BSOR reduction by NADPH (>1000 s(-1)) is faster than steady-state turnover (440 s(-1)) and has shown that BSOR reduction occurs in concert with NADPH oxidation with no indication of a Mo(V) intermediate species. Because no crystallographic structure is currently available for BSOR, a protein structure was modeled using the structures for R. sphaeroides dimethyl sulfoxide reductase, Rhodobacter capsulatus dimethyl sulfoxide reductase, and Shewanella massilia trimethylamine N-oxide reductase as the templates. A potential NADPH-binding site was identified and tested by site-directed mutagenesis of residues within the area. Mutation of Arg137 or Asp136 reduced the ability of NADPH to serve as the electron donor to BSOR, indicating that the NADPH-binding site in BSOR is located in the active-site funnel of the putative structure where it can directly reduce the Mo center. Along with kinetic and spectroscopic data, the location of this binding site supports a direct hydride transfer mechanism for NADPH reduction of BSOR.     

Kinetic and mechanistic properties of biotin sulfoxide reductase

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase catalyzes the reduction of d-biotin d-sulfoxide (BSO) to biotin. Initial rate studies of the homogeneous recombinant enzyme, expressed in Escherichia coli, have demonstrated that the purified protein utilizes NADPH as a facile electron donor in the absence of any additional auxiliary proteins. We have previously shown [Pollock, V. V., and Barber, M. J. (1997) J. Biol. Chem. 272, 3355-3362] that, at pH 8 and in the presence of saturating concentrations of BSO, the enzyme exhibits, a marked preference for NADPH (k(cat,app) = 500 s(-1), K(m,app) = 269 microM, and k(cat,app)/K(m,app) = 1.86 x 10(6) M(-1) s(-1)) compared to NADH (k(cat,app) = 47 s(-1), K(m,app) = 394 microM, and k(cat,app)/K(m,app) = 1.19 x 10(5) M(-1) s(-1)). Production of biotin using NADPH as the electron donor was confirmed by both the disk biological assay and by reversed-phase HPLC analysis of the reaction products. The purified enzyme also utilized ferricyanide as an artificial electron acceptor, which effectively suppressed biotin sulfoxide reduction and biotin formation. Analysis of the enzyme isolated from tungsten-grown cells yielded decreased reduced methyl viologen:BSO reductase, NADPH:BSO reductase, and NADPH:FR activities, confirming that Mo is required for all activities. Kinetic analyses of substrate inhibition profiles revealed that the enzyme followed a Ping Pong Bi-Bi mechanism with both NADPH and BSO exhibiting double competitive substrate inhibition. Replots of the 1/v-axes intercepts of the parallel asymptotes obtained at several low concentrations of fixed substrate yielded a K(m) for BSO of 714 and 65 microM for NADPH. In contrast, utilizing NADH as an electron donor, the replots yielded a K(m) for BSO of 132 microM and 1.25 mM for NADH. Slope replots of data obtained at high concentrations of BSO yielded a K(i) for BSO of 6.10 mM and 900 microM for NADPH. Kinetic isotope studies utilizing stereospecifically deuterated NADPD indicated that BSO reductase uses specifically the 4R-hydrogen of the nicotinamide ring. Cyanide inhibited NADPH:BSO and NADPH:FR activities in a reversible manner while diethylpyrocarbonate treatment resulted in complete irreversible inactivation of the enzyme concomitant with molybdenum cofactor release, indicating that histidine residues are involved in cofactor-binding.     

Structure of the molybdenum site of Rhodobacter sphaeroides biotin sulfoxide reductase

Conditions for heterologous expression of Rhodobacter sphaeroides biotin sulfoxide reductase in Escherichia coli were modified, resulting in a significant improvement in the yield of recombinant enzyme and enabling structural studies of the molybdenum center. Quantitation of the guanine and the molybdenum as compared to that found in R. sphaeroides DMSO reductase demonstrated the presence of the bis(MGD)molybdenum cofactor. UV-visible absorption spectra were obtained for the oxidized, NADPH-reduced, and dithionite-reduced enzyme. EPR spectra were obtained for the Mo(V) state of the enzyme. X-ray absorption spectroscopy at the molybdenum K-edge has been used to probe the molybdenum coordination of the enzyme. The molybdenum site of the oxidized protein possesses a Mo(VI) mono-oxo site (Mo=O at 1.70 A) with additional coordination by approximately four thiolate ligands at 2.41 A and probably one oxygen or nitrogen at 1.95 A. The NADPH- and dithionite-reduced Mo(IV) forms of the enzyme are des-oxo molybdenum sites with approximately four thiolates at 2.33 A and two different Mo-O/N ligands at 2.19 and 1.94 A.     

An active site tyrosine influences the ability of the dimethyl sulfoxide reductase family of molybdopterin enzymes to reduce S-oxides

Dimethyl sulfoxide reductase (DMSOR), trimethylamine-N-oxide reductase (TMAOR), and biotin sulfoxide reductase (BSOR) are members of a class of bacterial oxotransferases that contain the bis(molybdopterin guanine dinucleotide)molybdenum cofactor. The presence of a Tyr residue in the active site of DMSOR and BSOR that is missing in TMAOR has been implicated in the inability of TMAOR, unlike DMSOR and BSOR, to utilize S-oxides. To test this hypothesis, Escherichia coli TMAOR was cloned and expressed at high levels, and site-directed mutagenesis was utilized to generate the Tyr-114 --> Ala and Phe variants of Rhodobacter sphaeroides DMSOR and insert a Tyr residue into the equivalent position in TMAOR. Although all of the mutants turn over in a manner similar to their respective wild-type enzymes, mutation of Tyr-114 in DMSOR results in a decreased specificity for S-oxides and an increased specificity for trimethylamine-N-oxide (Me(3)NO), with a greater change observed for DMSOR-Y114A. Insertion of a Tyr into TMAOR results in a decreased preference for Me(3)NO relative to dimethyl sulfoxide. Kinetic analysis and UV-visible absorption spectra indicate that the ability of DMSOR to be reduced by dimethyl sulfide is lost upon mutation of Tyr-114 and that TMAOR does not exhibit this activity even in the Tyr insertion mutant.     

Resonance Raman characterization of biotin sulfoxide reductase. Comparing oxomolybdenum enzymes in the ME(2)SO reductase family

Resonance Raman spectroscopy has been used to define active site structures for oxidized Mo(VI) and reduced Mo(IV) forms of recombinant Rhodobacter sphaeroides biotin sulfoxide reductase expressed in Escherichia coli. On the basis of (18)O/(16)O labeling studies involving water and the alternative substrate dimethyl sulfoxide and the close correspondence to the resonance Raman spectra previously reported for dimethyl sulfoxide reductase (Garton, S. D., Hilton, J., Oku, H., Crouse, B. R., Rajagopalan, K. V., and Johnson, M. K. (1997) J. Am. Chem. Soc. 119, 12906-12916), vibrational modes associated with a terminal oxo ligand and the two molybdopterin dithiolene ligands have been assigned. The results indicate that the enzyme cycles between mono-oxo-Mo(VI) and des-oxo-Mo(IV) forms with both molybdopterin dithiolene ligands remaining coordinated in both redox states. Direct evidence for an oxygen atom transfer mechanism is provided by (18)O/(16)O labeling studies, which show that the terminal oxo group at the molybdenum center is exchangeable with water during redox cycling and originates from the substrate in substrate-oxidized samples. Biotin sulfoxide reductase is not reduced by biotin or the nonphysiological products, dimethyl sulfide and trimethylamine. However, product-induced changes in the Mo=O stretching frequency provide direct evidence for a product-associated mono-oxo-Mo(VI) catalytic intermediate. The results indicate that biotin sulfoxide reductase is thermodynamically tuned to catalyze the reductase reaction, and a detailed catalytic mechanism is proposed.     

Identification of the iron-sulfur center of spinach ferredoxin-nitrite reductase as a tetranuclear center, and preliminary EPR studies of mechanism.

EPR spectroscopic and chemical analyses of spinach nitrite reductase show that the enzyme contains one reducible iron-sulfur center, and one site for binding either cyanide or nitrite, per siroheme. The heme is nearly all in the high spin ferric state in the enzyme as isolated. The extinction coefficient of the enzyme has been revised to E386 = 7.6 X 10(4) cm-1 (M heme)-1. The iron-sulfur center is reduced with difficulty by agents such as reduced methyl viologen (equilibrated with 1 atm of H2 at pH 7.7 in the presence of hydrogenase) or dithionite. Complexation of the enzyme with CO (a known ligand for nitrite reductase heme) markedly increases the reducibility of the iron-sulfur center. New chemical analyses and reinterpretation of previous data show that the enzyme contains 6 mol of iron and 4 mol of acid-labile S2-/mol of siroheme. The EPR spectrum of reduced nitrite reductase in 80% dimethyl sulfoxide establishes clearly that the enzyme contains a tetranuclear iron-sulfur (Fe4S4) center. The ferriheme and Fe4S4 centers are reduced at similar rates (k = 3 to 4 s-1) by dithionite. The dithionite-reduced Fe4S4 center is rapidly (k = 100 s-1) reoxidized by nitrite. These results indicate a role for the Fe4S4 center in catalysis.     

Electron transfer between reduced methyl viologen and oxidized glutathione: a new assay of Saccharomyces cerevisiae glutathione reductase

Pure glutathione reductase from Saccharomyces cerevisiae catalyzed under anaerobic conditions the enzymatic reduction of GSSG using electrochemically reduced methyl viologen as electron donor. The new assay was completely dependent on the amount of active enzyme present, and involved the formation of 1 mol GSH per mole of reduced methyl viologen consumed. The enzyme followed a standard Michaelis-Menten kinetics; a Km = 230 microM for reduced methyl viologen and a turnover number of 969 mumol GSSG reduced per minute per micromole enzyme were determined. The enzymatic activity seemed to depend on the redox potential, showing half-maximal activity at -0.407 V. The enzyme was quite specific: the activity using reduced benzyl viologen as electron donor was just 1.5% of that obtained with reduced methyl viologen at the same concentration and potential. Glutathione reductase was totally inactivated after a brief anaerobic exposure with reduced methyl viologen in the absence of GSSG; a partial reactivation was observed following addition of glutathione disulfide. No inhibition of the methyl viologen-dependent activity was observed in the presence of 2',5'-ADP or 2'-P-5'-ADP-ribose, two NADP(H) analogs, at concentrations which drastically inhibited the NADPH-dependent activity, thus suggesting that the reduced viologen does not interact with the pyridine nucleotide-binding site.     

Nitrite and hydroxylamine reduction in higher plants. Fractionation, electron donor and substrate specificity of leaf enzymes, principally from vegetable marrow (Cucurbita pepo L.)

Nitrite reductase was purified between 760- and 1300-fold from vegetable marrow (Cucurbita pepo L.) and residual hydroxylamine reductase activity was low or negligible by comparison. With ferredoxin as electron donor, nitrite loss and ammonia formation at pH7.5 were stoicheiometrically equivalent. Crude nitrite reductase preparations showed negligible activity with NADPH as electron donor maintained in the reduced state by glucose 6-phosphate, whereas by comparison, activity was high when either ferredoxin or benzyl viologen were also present and reduced by the NADPH-glucose 6-phosphate system, whereas FMNH(2) produced variable and relatively low activity under the same conditions. At pH values below 7, non-enzymic reactions occurred between reduced benzyl viologen and nitrite, and intermediate reduction products were inferred to be produced instead of ammonia. Activity with ferredoxin (0.1mm), reduced by chloroplast grana in the light, was 25 times that produced with ferredoxin (40mum) reduced with NADPH and glucose 6-phosphate. For an approximate molecular weight 61000-63000 derived by chromatography on Sephadex G-100 and G-200, and a specific activity of 46mumol of nitrite reduced/min per mg of protein with light and chloroplast grana, a minimum turnover number of 3x10(3)mol of nitrite reduced/min per mol of enzyme was found. Two hydroxylamine reductases were separated on Sephadex gels. One (HR1) was initially associated with nitrite reductase during gel filtration but disappeared during later fractionation. This HR1 fraction showed nearly comparable activity with reduced benzyl viologen, ferredoxin or FMNH(2). The other (HR2), of molecular weight approx. 35000, reacted with reduced benzyl viologen but showed negligible activity with ferredoxin or NADPH. Activity with FMNH(2) was associated with an irregular trailing boundary during gel filtration, with much diminished activity in the HR2 region. Activity with NADPH was about 30% of that with FMNH(2), reduced benzyl viologen or ferredoxin and was considered to reside in fraction HR1. Hydroxylamine yielded ammonia under all assay conditions. No activity with hyponitrite or sulphite was observed with reduced benzyl viologen as electron donor in either the nitrite reductase or the hydroxylamine reductase systems, but pyruvic oxime produced about 4% of the activity of hydroxylamine.     

Superoxide production by dinitrophenyl-derivatized thioredoxin reductase--a model for the mechanism and correlation to immunostimulation by dinitrohalobenzenes

Mammalian thioredoxin reductase catalyzes NADPH dependent reduction of a wide variety of substrates and plays a central role in redox regulation and antioxidant defence. Recently the enzyme was discovered to be a selenoprotein with a catalytically active penultimate selenocysteine residue. Dinitrohalobenzenes irreversibly inhibit the enzyme with a concomitant induction of an NADPH oxidase activity, producing superoxide. A model explaining the reactivity of dinitrohalobenzenes with thioredoxin reductase is presented, involving dinitrophenyl-derivatization of both the selenocysteine residue and its neighboring cysteine residue, reduction by NADPH of the enzyme-bound flavin in dinitrophenyl-alkylated enzyme (dnp-TrxR), followed by two consecutive one-electron transfers from the flavin to nitro groups of the dnp-moieties in dnp-TrxR, forming nitro anion radicals. The nitro radicals react with oxygen to form superoxide, again generating dnp-TrxR with an oxidized flavin, which may then follow another cycle of NADPH-dependent superoxide production. Dinitrohalobenzene compounds are well known for their immunostimulatory properties. Here it is proposed that the inflammatory components of this immunostimulation can be mediated by interaction with the thioredoxin system, via effects on cell function by superoxide production, oxidative stress and increased extracellular levels of thioredoxin.     

Cysteine-125 is the catalytic residue of Saccharomyces cerevisiae free methionine-R-sulfoxide reductase

Free methionine-R-sulfoxide reductase (fRMsr) is a new type of methionine sulfoxide reductase that catalyzes the reduction of free methionine-R-sulfoxide to methionine. This enzyme cannot reduce oxidized methionine residues in proteins. While three Cys residues, Cys-91, Cys-101 and Cys-125, have been demonstrated to be involved in the catalysis by Saccharomyces cerevisiae fRMsr, their specific functions have not been fully established. In this work, we performed in vivo growth complementation experiments using S. cerevisiae cells lacking all three known methionine sulfoxide reductases. Cells containing a C125S construct, in which Cys-125 in fRMsr was replaced with Ser, did not grow in methionine sulfoxide medium, whereas cells containing C91S, C101S, or C91/101S constructs could grow in this medium. In addition, when assayed with thioredoxin and glutaredoxin reduction systems, the C125S form was inactive, whereas C91S and C101S had 1-2% and 9-10%, respectively, of the activity of the wild-type fRMsr. These data show that Cys-125 is the catalytic residue in fRMsr.     

Site-directed mutagenesis of dimethyl sulfoxide reductase from Rhodobacter capsulatus: characterization of a Y114 --> F mutant

A system for expressing site-directed mutants of the molybdenum enzyme dimethyl sulfoxide reductase from Rhodobacter capsulatus in the natural host was constructed. This system was used to generate and express dimethyl sulfoxide reductase with a Y114F mutation. The Y114F mutant had an increased k(cat) and increased K(m) toward both dimethyl sulfoxide and trimethylamine N-oxide compared to the native enzyme, and the value of k(cat)/K(m) was lower for both substrates in the mutant enzyme. The Y114F mutant, as isolated, was able to oxidize dimethyl sulfide with phenazine ethosulfate as the electron acceptor but with a lower k(cat) than that of the native enzyme. The pH optimum of dimethyl sulfide:acceptor oxidoreductase activity in the Y114F mutant was shown to be shifted by +1 pH unit compared to the native enzyme. The Y114F mutant did not form a pink complex with dimethyl sulfide, which is characteristic of the native enzyme. The mutant enzyme showed a large increase in the K(d) for DMS. Direct electrochemistry showed that the Mo(V)/Mo(IV) couple was unaffected by the Y114F mutant, but the midpoint potential of the Mo(VI)/Mo(V) couple was raised by about 50 mV. These data confirm that the Y114 residue plays a critical role in oxidation-reduction processes at the molybdenum active site and in oxygen atom transfer associated with sulfoxide reduction.     

Human liver aldehyde reductase: pH dependence of steady-state kinetic parameters.

The pH dependence of steady-state parameters for aldehyde reduction and alcohol oxidation were determined in the human liver aldehyde reductase reaction. The maximum velocity of aldehyde reduction with NADPH or 3-acetyl pyridine adenine dinucleotide phosphate (3-APADPH) was pH independent at low pH but decreased at high pH with a pK of 8.9-9.6. The V/K for both nucleotides decreased below a pK of 5.7-6.2, as did the pKi of competitive inhibitors NADP and ATP-ribose, suggesting that the 2'-phosphate of the nucleotide has to be deprotonated for binding to the enzyme. The pK of the 2'-phosphate of NADPH appears to be perturbed in the ternary complexes to 5.2-5.4. The V/K for NADPH, the V/K for 3-APADPH, and the pKi of ATP-ribose also decreased above a pK of 9-10, suggesting interaction of the 2'-phosphate of the nucleotide with a protonated base, perhaps lysine. Since protonation of a residue with a pK of 8 (evident in V/K for DL-glyceraldehyde and V/K for L-gulonate versus pH profiles) appears to be essential for aldehyde reduction, and deprotonation for alcohol oxidation, this residue appears to act as a general acid-base catalyst. An additional anion binding site with a pK of 9.94 facilitates the binding of carboxylic substrates such as D-glucuronate. With NADPH as the coenzyme the primary deuterium isotope effects on V and V/K for NADPH were close to unity and pH independent, suggesting that the hydride transfer step is not rate determining over the experimental pH range. With 3-APADPH as the coenzyme, the maximum velocity, relative to NADPH was three- to four-fold lower. Isotope effects on V, V/K for 3-APADPH, and V/K for D-glucuronate were pH independent and equal to 2.2-2.8, indicating that the chemical step of the reaction is relatively insensitive to pH. These data suggest that substrates bind to both the protonated and the deprotonated forms of the enzyme, though only the protonated enzyme catalyzes aldehyde reduction and the deprotonated enzyme catalyzes alcohol oxidation. On the basis of these results a scheme for the chemical mechanism of aldehyde reductase is postulated.     

Investigations into the role of the plastidial peptide methionine sulfoxide reductase in response to oxidative stress in Arabidopsis

Peptidyl Met residues are readily oxidized by reactive oxygen species to form Met sulfoxide. The enzyme peptide Met sulfoxide reductase (PMSR) catalyzes the reduction of Met sulfoxides back to Met. In doing so, PMSR is proposed to act as a last-chance antioxidant, repairing proteins damaged from oxidative stress. To assess the role of this enzyme in plants, we generated multiple transgenic lines with altered expression levels of the plastid form of PMSR (PMSR4). In transgenic plants, PMSR4 expression ranged from 95% to 40% (antisense) and more than 600% (overexpressing lines) of wild-type plants. Under optimal growing conditions, there is no effect of the transgene on the phenotype of the plants. When exposed to different oxidative stress conditions-methyl viologen, ozone, and high light-differences were observed in the rate of photosynthesis, the maximum quantum yield (Fv/Fm ratio), and the Met sulfoxide content of the isolated chloroplast. Plants that overexpressed PMSR4 were more resistant to oxidative damage localized in the chloroplast, and plants that underexpressed PMSR4 were more susceptible. The Met sulfoxide levels in proteins of the soluble fraction of chloroplasts were increased by methyl viologen and ozone, but not by high-light treatment. Under stress conditions, the overexpression of PMSR4 lowered the sulfoxide content and underexpression resulted in an overall increase in content.     

Some properties of an NADH-benzyl viologen reductase from azotobacter vinelandii

1. NADH-benzyl viologen reductase, solubilized by acetone extraction, was purified about 10-fold from small particles of Azotobacter vinelandii.

2. The purified enzyme preparation was free from hydrogenase activity. Either NADH or NADPH served as an electron donor for the reduction of benzyl viologen. This reaction is reversible.

3. The essential thiol groups of the enzyme are protected since they do not react with N-ethylmaleimide and p-chloromercuribenzoate inhibits only after it has been preincubated with the enzyme.     

Interaction of NADP(H) with oxidized and reduced P450 reductase during catalysis. Studies with nucleotide analogues

Previous studies have shown that the interaction of P450 reductase with bound NADP(H) is essential to ensure fast electron transfer through the two flavin cofactors. In this study we investigated in detail the interaction of the house fly flavoprotein with NADP(H) and a number of nucleotide analogues. 1,4,5,6-Tetrahydro-NADP, an analogue of NADPH, was used to characterize the interaction of P450 reductase with the reduced nucleotide. This analogue is inactive as electron donor, but its binding affinity and rate constant of release are very close to those for NADPH. The 2'-phosphate contributes about 5 kcal/mol of the binding energy of NADP(H). Oxidized nicotinamide does not interact with the oxidized flavoprotein, while reduced nicotinamide contributes 1.3 kcal/mol of the binding energy. Oxidized P450 reductase binds NADPH with a K(d) of 0.3 microM, while the affinity of the reduced enzyme is considerably lower, K(d) = 1.9 microM. P450 reductase catalyzes a transhydrogenase reaction between NADPH and oxidized nucleotides, such as thionicotinamide-NADP(+), acetylpyridine-NADP(+), or [(3)H]NADP(+). The reverse reaction, reduction of [(3)H]NADP(+) by the reduced analogues, is also catalyzed by P450 reductase. We define the mechanism of the transhydrogenase reaction as follows: NADPH binding, hydride ion transfer, and release of the NADP(+) formed. An NADP(+) or its analogue binds to the two-electron-reduced flavoprotein, and the electron-transfer steps reverse to transfer hydride ion to the oxidized nucleotide, which is released. Measurements of the flavin semiquinone content, rate constant for NADPH release, and transhydrogenase turnover rates allowed us to estimate the steady-state distribution of P450 reductase species during catalysis, and to calculate equilibrium constants for the interconversion of catalytic intermediates. Our results demonstrate that equilibrium redox potentials of the flavin cofactors are not the sole factor governing rapid electron transfer during catalysis, but conformational changes must be considered to understand P450 reductase catalysis.     

Molybdenum cofactor requirement for biotin sulfoxide reduction in Escherichia coli.

The bisC gene of Escherichia coli is tentatively identified as the structural gene for biotin sulfoxide reductase by the isolation of bisC(Ts) mutants that make thermolabile enzyme. The products of four other E. coli genes (chlA, chlB, chlE and chlG) are also needed for enzymatic activity. Mutations previously assigned to the bisA, bisB, and bisD genes belong to genes chlA, chlE, and chlG, respectively. The biotin sulfoxide reductase deficiency of a chlG, mutant is partially reversed by the addition of 10 mM molybdate to the growth medium. Mutational inactivation of the chlD gene reduces the specific activity of biotin sulfoxide reductase about twofold. This effect is reversed by the addition of 1 mM molybdate to the growth medium. The specific activity of biotin sulfoxide reductase is decreased about 30-fold by the presence of tungstate in the growth medium, an effect that has been observed previously with nitrate reductase and other molybdoenzymes. The specific activity of biotin sulfoxide reductase is not elevated in a lysate prepared by derepressing a lambda cI857 chlG prophage. Whereas biotin sulfoxide reductase prepared by sonic extraction of growing cells is almost completely dependent on the presence of a small heat-stable protein resembling thioredoxin, much of the enzyme obtained from lysates of thermoinduced lambda cI857 lysogens does not require this factor.     

Mechanism of cytochrome P450 reductase from the house fly: evidence for an FMN semiquinone as electron donor.

The interaction of recombinant house fly (Musca domestica) P450 reductase with NADPH and the role of the FMN semiquinone in reducing cytochrome c have been investigated. House fly P450 reductase can rapidly oxidize only one molecule of NADPH, whereas the rate of oxidation of a second molecule of NADPH is too slow to account for the observed rates of catalysis. This demonstrates that house fly P450 reductase does not require a priming reaction with NADPH for catalysis. Kinetics of cytochrome c reduction and EPR spectroscopy revealed that the enzyme forms two types of neutral FMN semiquinone. One serves as the catalytic intermediate of cytochrome c reduction, and another one is an 'airstable' semiquinone, which reduces cytochrome c 3000 times more slowly. The results show that the reduction state of the house fly P450 reductase during catalysis cycles in a 0-2-1-0 sequence.     

Acid-base catalysis in the mechanism of thioredoxin reductase from Drosophila melanogaster

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin (Trx) by NADPH. Like other members of the pyridine nucleotide-disulfide oxidoreductase enzyme family, the enzyme from Drosophila melanogaster is a homodimer, and each catalytically active unit consists of three redox centers: FAD and an N-terminal Cys-57/Cys-62 redox-active disulfide from one monomer and a Cys-489'/Cys-490' C-terminal redox-active disulfide from the second monomer. Because dipteran insects such as D. melanogaster lack glutathione reductase, thioredoxin reductase (DmTrxR) is particularly important; in addition to its normal functions, it also reduces GSSG for antioxidant protection. DmTrxR, used as a model for the enzyme from the malaria vector, Anopheles gambiae, has been shown to cycle in catalysis between the two-electron and four-electron reduced states, EH2 and EH4 [Bauer, H. et al. (2003) J. Biol. Chem. 278, 33020-33028]. His-464' acts as an acid-base catalyst of the dithiol-disulfide interchange reactions required in catalysis. The H464'Q enzyme has only 2% of the wild-type activity, emphasizing the importance of this residue. The pH dependence of Vmax for wild-type DmTrxR has pKa values of 6.4 and 9.3 on the DmTrxR-DmTrx-2 complex, whereas H464'Q DmTrxR only has an observable pKa at 6.4, indicating that the pKa at pH 9.3 is contributed mainly by His-464'. The pKa at pH 6.4 has been assigned to Cys-57 and Cys-490'; the thiolate on Cys-490' is the nucleophile in the reduction of Trx. In contrast to wild-type DmTrxR, H464'Q DmTrxR does not stabilize a thiolate-FAD charge-transfer complex in the presence of excess NADPH. The rates of steps in both the reductive and the oxidative half-reactions are markedly diminished in H464'Q DmTrxR as compared to those of wild-type enzyme, indicating that His-464' is involved in both half-reactions.     

Cobalt hexaamine mediated electrocatalytic voltammetry of dimethyl sulfoxide reductase: driving force effects on catalysis

The bacterial molybdoenzyme dimethyl sulfoxide (DMSO) reductase from Rhodobacter capsulatus catalyzes the reduction of DMSO to dimethyl sulfide in anaerobic respiration. In its native state, DMSO reductase is reduced to its active state by a pentaheme cytochrome (DorC). Alternatively, we show that DMSO reductase catalysis may be driven electrochemically using a series of homologous coordination compounds as mediating synthetic electron donors. All mediators are macrocyclic hexaaminecobalt(II) complexes in their active form, differing principally in their redox potentials over a range of about 250 mV. Thus, each complex presents a different reductive driving force to DMSO reductase and this leads to pronounced differences in the electrocatalytic behavior as measured by cyclic voltammetry. Digital simulation of the experimental voltammetry enables the critical features of the catalytic cycle to be extracted.