The first non-turnover voltammetric response from a molybdenum enzyme: direct electrochemistry of dimethylsulfoxide reductase from Rhodobacter capsulatus

The first direct voltammetric response from a molybdenum enzyme under non-turnover conditions is reported. Cyclic voltammetry of dimethylsulfoxide reductase from Rhodobacter capsulatus reveals a reversible Mo(VI/V) response at +161 mV followed by a reversible Mo(V/IV) response at -102 mV versus NHE at pH 8. The higher potential couple exhibits a pH dependence consistent with protonation upon reduction to the Mo(V) state and we have determined the p K(a) for this semi-reduced species to be 9.0. The lower potential couple is pH independent within the range 5相似文献   

Resonance Raman studies on xanthine oxidase: observation of Mo<Superscript>VI</Superscript>-ligand vibrations

Resonance Raman spectra were investigated for the sulfo and desulfo forms of cow's milk xanthine oxidase, with various visible excitation lines between 400 and 650 nm, and Mo(VI)-ligand vibrations were observed for the first time. The Mo(VI)=S stretch was identified at 474 and 462 cm(-1 )for the (32)S- and (34)S-sulfo forms, respectively, but was absent in the reduced state and in the desulfo form. The Mo(VI)=O stretch was weakly observed at 899 cm(-1 )for the sulfo form and shifted to 892 cm(-1) with very weak intensity for the dioxo desulfo form. In measurements of an excitation profile, the two bands at 474 and 899 cm(-1) showed maximum intensity at similar excitation wavelengths, suggesting that the Raman intensity of the metal-ligand modes is due to the Mo(VI)<--S charge transfer transition, and that this is the origin of the intrinsically weak features of the Mo(VI)-ligand Raman bands. When the sulfo form was regenerated from the desulfo form, the 899 cm(-1) band reappeared. However, the band at 899 cm(-1) showed no frequency shift when regeneration was conducted in H(2)(18)O, or after several turnovers in the presence of xanthine in H(2)(18)O. When the sulfo form was reduced and reoxidized in H(2)(18)O buffer, the 899 cm(-1) band reappeared without any frequency shift. These observations suggest that the oxo oxygen in the Mo center of xanthine oxidase is not labile. Low-frequency vibrations of the Mo center were observed together with those of the Fe(2)S(2) center with some overlaps, while FAD modes were observed clearly. The absence of dithiolene modes in XO is in contrast to the Mo(VI) centers of DMSO reductase and sulfite oxidase.     

Reactions of dimethylsulfoxide reductase in the presence of dimethyl sulfide and the structure of the dimethyl sulfide-modified enzyme

The bis-molybdopterin enzyme dimethylsulfoxide reductase (DMSOR) from Rhodobacter capsulatus catalyzes the conversion of dimethyl sulfoxide (DMSO) to dimethyl sulfide (DMS), reversibly, in the presence of suitable e(-)-donors or e(-)-acceptors. The catalytically significant intermediate formed by reaction of DMSOR with DMS ('the DMS species') and a damaged enzyme form derived by reaction of the latter with O(2) (DMS-modified enzyme, DMSOR(mod)D) have been investigated. Evidence is presented that Mo in the DMS species is not, as widely assumed, Mo(IV). Formation of the DMS species is reversed on removing DMS or by addition of an excess of DMSO. Equilibrium constants for the competing reactions of DMS and DMSO with the oxidized enzyme (K(d) = 0.07 +/- 0.01 and 21 +/- 5 mM, respectively) that control these processes indicate formation of the DMS species occurs at a redox potential that is 80 mV higher than that required, according to the literature, for reduction of Mo(VI) to Mo(IV) in the free enzyme. Specificity studies show that with dimethyl selenide, DMSOR yields a species analogous to the DMS species but with the 550 nm peak blue-shifted by 27 nm. It is concluded from published redox potential data that this band is due to metal-to-ligand charge transfer from Mo(V) to the chalcogenide. Since the DMS species gives no EPR signal in the normal or parallel mode, a free radical is presumed to be in close proximity to the metal, most likely on the S. The species is thus formulated as Mo(V)-O-S(*)Me(2). Existing X-ray crystallographic and Raman data are consistent with this structure. Furthermore, 1e(-) oxidation of the DMS species with phenazine ethosulfate yields a Mo(V) form without an -OH ligand, since its EPR signal shows no proton splittings. This form presumably arises via dissociation of DMSO. The structure of DMSOR(mod)D has been determined by X-ray crystallography. All four thiolate ligands and Ogamma of serine-147 remain coordinated to Mo, but there are no terminal oxygen ligands and Mo is Mo(VI). Thus, it is a dead-end species, neither oxo group acceptance nor e(-)-donation being possible. O(2)-dependent formation of DMSOR(mod)D represents noncatalytic breakdown of the DMS species by a pathway alternative to that in turnover, with oxidation to Mo(VI) presumably preceding product release. Steps in the forward and backward catalytic cycles are discussed in relation to earlier stopped-flow data. The finding that in the back-assay the Mo(IV) state may at least in part be by-passed via two successive 1e(-) reactions of the DMS species with the e(-)-acceptor, may have implications in relation to the existence of separate molybdopterin enzymes catalyzing DMSO reduction and DMS oxidation, respectively.     

Spectroscopic studies of the molybdenum-containing dimethyl sulfoxide reductase from Rhodobacter sphaeroides f. sp. denitrificans.

Absorption and EPR spectroscopic properties of purified dimethyl sulfoxide (Me2SO) reductase from Rhodobacter sphaeroides f. sp. denitrificans have been examined. The absence of prosthetic groups other than the molybdenum center in the enzyme has made it possible to study its absorption properties. The enzyme displays multiple absorbance peaks in both the oxidized and the dithionite-reduced forms. The oxidized enzyme has absorbance peaks at 280, 350, 470, 550, and 720 nm while the dithionite-reduced enzyme has peaks at 280, 374, and 645 nm with a shoulder at 430 nm. A comparison of the absorbance spectrum of oxidized Me2SO reductase with that of the molybdenum fragment of rat liver sulfite oxidase shows that the 350 and 470 peaks are common to both proteins. EPR studies of the Mo(V) form of Me2SO reductase show a rhombic signal with g1 = 1.988, g2 = 1.977, g3 = 1.961, and g(ave) = 1.975. The signal shows evidence of coupling to an exchangeable proton with A1 = 1.05, A2 = 1.13, A3 = 0.98, and Aave = 1.05 millitesla. These parameters are similar to those of other Mo enzymes, however, the epr signal of this enzyme differs from those of other Mo hydroxylases in showing only a slight sensitivity to pH and no detectable anion effect. EPR potentiometric titrations of Me2SO reductase gave midpoint potentials of +144 mV for the Mo(VI)/Mo(V) couple and +160 mV for the Mo(V)/Mo(IV) couple at room temperature and +141 mV for the Mo(VI)/Mo(V) couple and +200 mV for the Mo(V)/Mo(IV) couple at 173 K.     

Synthesis and reactivity studies of model complexes for molybdopterin-dependent enzymes

The molybdenum cofactor (Moco)-containing enzymes are divided into three classes that are named after prototypical members of each family, viz. sulfite oxidase, DMSO reductase and xanthine oxidase. Functional or structural models have been prepared for these three prototypical enzymes: (i) The complex [MoO2(mnt)2]2- (mnt2- = 1,2-dicyanoethylenedithiolate) has been found to be able to oxidize hydrogen sulfite to HSO4- and is thus a functional model of sulfite oxidase. Kinetic and computational studies indicate that the reaction proceeds via attack of the substrate at one of the oxo ligands of the complex, rather than at the metal. (ii) The coordination geometries of the mono-oxo [Mo(VI)(O-Ser)(S2)2] entity (S2 = dithiolene moiety of molybdopterin) found in the crystal structure of R. sphaeroides DMSO reductase and the corresponding des-oxo Mo(IV) unit have been reproduced in the complexes [M(VI)O(OSiR3)(bdt)2] and [M(VI)O(OSiR3)(bdt)2] (M = Mo,W; bdt = benzene dithiolate). (iii) A facile route has been developed for the preparation of complexes containing a cis-Mo(VI)OS molybdenum oxo, sulfido moiety similar to that detected in the oxidized form of xanthine oxidase.     

X-ray absorption spectroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus

Mo K-edge X-ray absorption spectroscopy (XAS) has been used to probe the environment of Mo in dimethylsulfoxide (DMSO) reductase from Rhodobacter capsulatus in concert with protein crystallographic studies. The oxidised (Mo^VI) protein has been investigated in solution at 77?K; the Mo K-edge position (20006.4?eV) is consistent with the presence of Mo^VI and, in agreement with the protein crystallographic results, the extended X-ray absorption fine structure (EXAFS) is also consistent with a seven-coordinate site. The site is composed of one oxo-group (Mo=O 1.71?Å), four S atoms (considered to arise from the dithiolene groups of the two molybdopterins, two at 2.32?Å and two at 2.47?Å, and two O atoms, one at 1.92?Å (considered to be H-bonded to Trp 116) and one at 2.27?Å (considered to arise from Ser 147). The Mo K-edge XAS recorded for single crystals of oxidised (Mo^VI) DMSO reductase at 77?K showed a close correspondence to the data for the frozen solution but had an inferior signal:noise ratio. The dithionite-reduced form of the enzyme and a unique form of the enzyme produced by the addition of dimethylsulfide (DMS) to the oxidised (Mo^VI) enzyme have essentially identical energies for the Mo K-edge, at 20004.4?eV and 20004.5?eV, respectively; these values, together with the lack of a significant presence of Mo^V in the samples as monitored by EPR spectroscopy, are taken to indicate the presence of Mo^IV. For the dithionite-reduced sample, the Mo K-edge EXAFS indicates a coordination environment for Mo of two O atoms, one at 2.05?Å and one at 2.51?Å, and four S atoms at 2.36?Å. The coordination environment of the Mo in the DMS-reduced form of the enzyme involves three O atoms, one at 1.69?Å, one at 1.91?Å and one at 2.11?Å, plus four S atoms, two at 2.28?Å and two at 2.37?Å. The EXAFS and the protein crystallographic results for the DMS-reduced form of the enzyme are consistent with the formation of the substrate, DMSO, bound to Mo^IV with an Mo-O bond of length 1.92?Å.     

Resonance Raman as a direct probe for the catalytic mechanism of molybdenum oxotransferases

 Recent studies of human sulfite oxidase and Rhodobacter sphaeroides DMSO reductase have demonstrated the ability of resonance Raman to probe in detail the coordination environment of the Mo active sites in oxotransferases via Mo=O, Mo-S(dithiolene), Mo-S(Cys) or Mo-O(Ser), dithiolene chelate ring and bound substrate vibrations. Furthermore, the ability to monitor the catalytically exchangeable oxo group via isotopic labeling affords direct mechanistic information and structures for the catalytically competent Mo(IV) and Mo(VI) species. The results clearly demonstrate that sulfite oxidase cycles between cis–di-oxo-Mo(VI) and mono-oxo-Mo(IV) states during catalytic turnover, whereas DMSO reductase cycles between mono-oxo-Mo(VI) and des-oxo-Mo(IV) states. In the case of DMSO reductase, ¹⁸O-labeling experiments have provided the first direct evidence for an oxygen atom transfer mechanism involving an Mo=O species. Of particular importance is that the active-site structures and detailed mechanism of DMSO reductase in solution, as determined by resonance Raman spectroscopy, are quite different to those reported or deduced in the three X-ray crystallographic studies of DMSO reductases from Rhodobacter species. Received: 16 June 1997 / Accepted: 20 August 1997     

Oxidation-reduction midpoint potentials of the molybdenum center in spinach NADH:nitrate reductase

Oxidation-reduction midpoint potentials for the molybdenum center in assimilatory NADH:nitrate reductase isolated from spinach (Spinacia oleracea) have been determined at pH 7.0 in the presence of dye mediators using EPR spectroscopy to monitor formation of Mo(V). Values for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples were determined to be -8 and -42 mV, respectively.     

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.     

Models for molybdenum coordination during the catalytic cycle of periplasmic nitrate reductase from Paracoccus denitrificans derived from EPR and EXAFS spectroscopy.

The periplasmic nitrate reductase from Paracoccus denitrificans is a soluble two-subunit enzyme which binds two hemes (c-type), a [4Fe-4S] center, and a bis molybdopterin guanine dinucleotide cofactor (bis-MGD). A catalytic cycle for this enzyme is presented based on a study of these redox centers using electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) spectroscopies. The Mo(V) EPR signal of resting NAP (High g [resting]) has g(av) = 1.9898 is rhombic, exhibits low anisotropy, and is split by two weakly interacting protons which are not solvent-exchangeable. Addition of exogenous ligands to this resting state (e.g., nitrate, nitrite, azide) did not change the form of the signal. A distinct form of the High g Mo(V) signal, which has slightly lower anisotropy and higher rhombicity, was trapped during turnover of nitrate and may represent a catalytically relevant Mo(V) intermediate (High g [nitrate]). Mo K-edge EXAFS analysis was undertaken on the ferricyanide oxidized enzyme, a reduced sample frozen within 10 min of dithionite addition, and a nitrate-reoxidized form of the enzyme. The oxidized enzyme was fitted best as a di-oxo Mo(VI) species with 5 sulfur ligands (4 at 2. 43 A and 1 at 2.82 A), and the reduced form was fitted best as a mono-oxo Mo(IV) species with 3 sulfur ligands at 2.35 A. The addition of nitrate to the reduced enzyme resulted in reoxidation to a di-oxo Mo(VI) species similar to the resting enzyme. Prolonged incubation of NAP with dithionite in the absence of nitrate (i.e., nonturnover conditions) resulted in the formation of a species with a Mo(V) EPR signal that is quite distinct from the High g family and which has a g(av) = 1.973 (Low g [unsplit]). This signal resembles those of the mono-MGD xanthine oxidase family and is proposed to arise from an inactive form of the nitrate reductase in which the Mo(V) form is only coordinated by the dithiolene of one MGD. In samples of NAP that had been reduced with dithionite, treated with azide or cyanide, and then reoxidized with ferricyanide, two Mo(V) signals were detected with g(av) elevated compared to the High g signals. Kinetic analysis demonstrated that azide and cyanide displayed competitive and noncompetitive inhibition, respectively. EXAFS analysis of azide-treated samples show improvement to the fit when two nitrogens are included in the molybdenum coordination sphere at 2.52 A, suggesting that azide binds directly to Mo(IV). Based on these spectroscopic and kinetic data, models for Mo coordination during turnover have been proposed.     

Oxidation-reduction potentials of flavin and Mo-pterin centers in assimilatory nitrate reductase: variation with pH

Potentiometric titrations of assimilatory nitrate reductase from Chlorella vulgaris were performed within the pH range 6.0-9.0. Mo(V) was measured by room temperature EPR spectroscopy while the reduction state of FAD was monitored by CD spectroscopy. Between pH 6 and 8.5, the line shape of the Mo(V) EPR signal was constant, exhibiting superhyperfine coupling to a single, exchangeable proton. Potentiometric titrations indicated the Em values for the Mo(VI)/Mo(V) (+61 mV, pH 6) and Mo(V)/Mo(IV) (+35 mV, pH 6) couples decreased with increasing pH by approximately -59 mV/pH unit, consistent with the uptake of a single proton upon reduction of Mo(VI) to Mo(V) and Mo(V) to Mo(IV). The pKa values for the dissociation of these redox-coupled protons appeared to lie outside the pH range studied: pKo(MoVI), pKo(MoV) less than 5.5; pKr(MoV), pKr(MoIV) greater than 9. The Em (n = 2) for FAD (-250 mV, pH 7) varied by approximately -30 mV/pH unit within the pH range 6.0-9.0. Low-temperature EPR potentiometry at the extreme pH values indicated less than 0.5% conversion of FAD to the semiquinone form at the midpoint of the titrations. In contrast, NADH-reduced enzyme exhibited approximately 3-5% of the FAD in the semiquinone form, present as the anionic (FAD.-) species, the spectrum characterized by a line width of 1.3 mT at both pH 6.0 and 9.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of the 100 kDa arsenite oxidase from Alcaligenes faecalis in two crystal forms at 1.64 A and 2.03 A

BACKGROUND: Arsenite oxidase from Alcaligenes faecalis NCIB 8687 is a molybdenum/iron protein involved in the detoxification of arsenic. It is induced by the presence of AsO(2-) (arsenite) and functions to oxidize As(III)O(2-), which binds to essential sulfhydryl groups of proteins and dithiols, to the relatively less toxic As(V)O(4)(3-) (arsenate) prior to methylation. RESULTS: Using a combination of multiple isomorphous replacement with anomalous scattering (MIRAS) and multiple-wavelength anomalous dispersion (MAD) methods, the crystal structure of arsenite oxidase was determined to 2.03 A in a P2(1) crystal form with two molecules in the asymmetric unit and to 1.64 A in a P1 crystal form with four molecules in the asymmetric unit. Arsenite oxidase consists of a large subunit of 825 residues and a small subunit of approximately 134 residues. The large subunit contains a Mo site, consisting of a Mo atom bound to two pterin cofactors, and a [3Fe-4S] cluster. The small subunit contains a Rieske-type [2Fe-2S] site. CONCLUSIONS: The large subunit of arsenite oxidase is similar to other members of the dimethylsulfoxide (DMSO) reductase family of molybdenum enzymes, particularly the dissimilatory periplasmic nitrate reductase from Desulfovibrio desulfuricans, but is unique in having no covalent bond between the polypeptide and the Mo atom. The small subunit has no counterpart among known Mo protein structures but is homologous to the Rieske [2Fe-2S] protein domain of the cytochrome bc(1) and cytochrome b(6)f complexes and to the Rieske domain of naphthalene 1,2-dioxygenase.     

Oxidation--reduction potentials of molybdenum and iron--sulphur centres in nitrate reductase from Escherichia coli.

The potentials of the couples Mo(IV)--(Mo(V) and Mo(V)--Mo(VI) in nitrate reductase from Escherichia coli K12 were measured as + 180 mV and + 220 mV respectively at pH 7.14. The potentials associated with two other e.p.r. signals, believed to be due to iron--sulphur centres, were measured as + 50 mV and + 80 mV.     

Hexavalent chromate reduction by alkaliphilic Amphibacillus sp. KSUCr3 is mediated by copper-dependent membrane-associated Cr(VI) reductase

The present study was aimed to localize and characterize hexavalent chromate [Cr(VI)] reductase activity of the extreme alkaliphilic Amphibacillus sp. KSUCr3 (optimal growth pH 10.5). The resting cells were able to reduce about 62 % of the toxic heavy metal Cr(VI) at initial concentration of 200 μM within 30 min. Cell permeabilization resulted in decrease of Cr(VI) reduction in comparison to untreated cells. Enzymatic assays of different sub-cellular fractions of Amphibacillus sp. KSUCr3 demonstrated that the Cr(VI) reductase was mainly associated with the membranous fraction and expressed constitutively. In vitro studies of the crude enzyme indicated that copper ion was essential for Cr(VI) reductase activity. In addition, Ca2? and Mn2? slightly stimulated the chromate reductase activity. Glucose was the best external electron donor, showing enhancement of the enzyme activity by about 3.5-fold. The K (m) and V (max) determined for chromate reductase activity in the membranous fraction were 23.8 μM Cr(VI) and 72 μmol/min/mg of protein, respectively. Cr(VI) reductase activity was maximum at 40 °C and pH 7.0 and it was significantly inhibited in the presence of disulfide reducers (2-mercaptoethanol), ion chelating agent (EDTA), and respiratory inhibitors (CN and Azide). Complete reduction of 100 and 200 μM of Cr(VI) by membrane associated enzyme were observed within 40 and 180 min, respectively. However, it should be noted that biochemical characterization has been done with crude enzyme only, and that final conclusion can only be drawn with the purified enzyme.     

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.     

Reactivity of potential anti-diabetic molybdenum(VI) complexes in biological media: a XANES spectroscopic study

The application of Mo(VI) complexes as anti-diabetic agents is a subject of considerable recent interest. The stability and speciation of [Mo(VI)O(4)](2-) and three analogs of known anti-diabetic V(IV) complexes ([Mo(VI)O(2)L(2)]; where LH=2,4-pentanedione, l-cysteine ethyl ester or N,N-diethyldithiocarbamic acid) in natural and simulated biological fluids (including blood and its components, cell culture media, and artificial digestion systems) were studied using MoK-edge XANES (X-ray absorption near-edge structure) spectroscopy of freeze-dried samples at 20K. All of the studied [MoO(2)L(2)] complexes decomposed extensively under simulated gastric and intestinal digestion conditions (3 h at 310 K), as well as in blood plasma or in cell culture medium (24 h at 310 K). The reaction products of [MoO(4)](2-) and [MoO(2)L(2)] with biological fluids could be satisfactorily modelled (using multiple linear regression analyses) as mixtures of tetrahedral and octahedral Mo(VI) species (with O-donor ligands) in various ratios, which were dependent on the nature of the medium rather than that of the initial Mo(VI) compounds. Red blood cells take up Mo(VI) predominantly in the form of [MoO(4)](2-). Implications of these results to the development of Mo(VI)-based anti-diabetics and to the mechanisms of natural uptake and metabolism of Mo(VI) are discussed.     

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.     

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.     

Active site heterogeneity in dimethyl sulfoxide reductase from Rhodobacter capsulatus revealed by Raman spectroscopy

Raman spectroscopy has been used to investigate the structure of the molybdenum cofactor in DMSO reductase from Rhodobacter capsulatus. Three oxidized forms of the enzyme, designated 'redox cycled', 'as prepared', and DMSOR(mod)D, have been studied using 752 nm laser excitation. In addition, two reduced forms of DMSO reductase, prepared either anaerobically using DMS or using dithionite, have been characterized. The 'redox cycled' form has a single band in the Mo=O stretching region at 865 cm(-1) consistent with other studies. This oxo ligand is found to be exchangeable directly with DMS(18)O or by redox cycling. Furthermore, deuteration experiments demonstrate that the oxo ligand in the oxidized enzyme has some hydroxo character, which is ascribed to a hydrogen bonding interaction with Trp 116. There is also evidence from the labeling studies for a modified dithiolene sulfur atom, which could be present as a sulfoxide. In addition to the 865 cm(-1) band, an extra band at 818 cm(-1) is observed in the Mo=O stretching region of the 'as prepared' enzyme which is not present in the 'redox cycled' enzyme. Based on the spectra of unlabeled and labeled DMS reduced enzyme, the band at 818 cm(-1) is assigned to the S=O stretch of a coordinated DMSO molecule. The DMSOR(mod)D form, identified by its characteristic Raman spectrum, is also present in the 'as prepared' enzyme preparation but not after redox cycling. The complex mixture of forms identified in the 'as prepared' enzyme reveals a substantial degree of active site heterogeneity in DMSO reductase.     

Heavy metal ions inhibit molybdoenzyme activity by binding to the dithiolene moiety of molybdopterin in Escherichia coli

Molybdenum insertion into the dithiolene group on the 6-alkyl side-chain of molybdopterin is a highly specific process that is catalysed by the MoeA and MogA proteins in Escherichia coli. Ligation of molybdate to molybdopterin generates the molybdenum cofactor, which can be inserted directly into molybdoenzymes binding the molybdopterin form of the molybdenum cofactor, or is further modified in bacteria to form the dinucleotide form of the molybdenum cofactor. The ability of various metals to bind tightly to sulfur-rich sites raised the question of whether other metal ions could be inserted in place of molybdenum at the dithiolene moiety of molybdopterin in molybdoenzymes. We used the heterologous expression systems of human sulfite oxidase and Rhodobacter sphaeroides dimethylsulfoxide reductase in E. coli to study the incorporation of different metal ions into the molybdopterin site of these enzymes. From the added metal-containing compounds Na(2)MoO(4), Na(2)WO(4), NaVO(3), Cu(NO(3))(2), CdSO(4) and NaAsO(2) during the growth of E. coli, only molybdate and tungstate were specifically inserted into sulfite oxidase and dimethylsulfoxide reductase. Other metals, such as copper, cadmium and arsenite, were nonspecifically inserted into sulfite oxidase, but not into dimethylsulfoxide reductase. We showed that metal insertion into molybdopterin occurs beyond the step of molybdopterin synthase and is independent of MoeA and MogA proteins. Our study shows that the activity of molybdoenzymes, such as sulfite oxidase, is inhibited by high concentrations of heavy metals in the cell, which will help to further the understanding of metal toxicity in E. coli.