Sphaeroides Dimethyl Sulfoxide Reductase Research Articles

X-ray Absorption Spectroscopy of a Quantitatively Mo(V) Dimethyl Sulfoxide Reductase Species

Molybdenum K-edge X-ray absorption spectroscopy (XAS) has been used to probe the structure of a Mo(V) species that has been suggested to be a catalytic intermediate in the reaction of dimethyl sulfoxide (DMSO) reductase with the alternative substrate trimethylamine N-oxide (Bennet et al. Eur. J. Biochem. 1994, 255, 321-331; Cobb et al. J. Biol. Chem. 2005, 280, 11007-11017; Mtei, et al. J. Am. Chem. Soc. 2011, 133, 9672-9774). The oxidized Mo(VI) state of DMSO reductase has previously been structurally characterized as being six coordinate, with four sulfurs from pyranopterin dithiolene molybdenum cofactors, a terminal oxygen ligand, and an additional oxygen coordination from a serine residue. We find the most plausible structure for the Mo(V) active site is a five-coordinate species with four sulfur donors from the two pyranopterin dithiolene ligands, with an average Mo-S bond-length of 2.35 Å, plus a single oxygen donor at 1.99 Å, very likely from an Mo-OH ligand. Our results thus suggest that the oxygen of the serine residue has dissociated from the metal ion, suggesting hitherto unsuspected flexibility of the active site, and calling into question whether this putative intermediate is catalytically relevant. The relevance to previous Mo(V) electron paramagnetic resonance and other spectroscopic studies on DMSO reductase is discussed. XAS of an extensively studied Mo(V) form of Rhodobacter sphaeroides DMSO reductase (the high-g split species) shows that previously suggested structures for the active site are likely incorrect.

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.

X-ray Absorption Spectroscopic Characterization of the Molybdenum Site of Escherichia coli Dimethyl Sulfoxide Reductase

Structural studies of dimethyl sulfoxide (DMSO) reductases were hampered by modification of the active site during purification. We report an X-ray absorption spectroscopic analysis of the molybdenum active site of Escherichia coli DMSO reductase contained within its native membranes. The enzyme in these preparations is expected to be very close to the form found in vivo. The oxidized active site was found to have four Mo-S ligands at 2.43 A, one Mo=O at 1.71 A, and a longer Mo-O at 1.90 A. We conclude that the oxidized enzyme is a monooxomolybdenum(VI) species coordinated by two molybdopterin dithiolenes and a serine. The bond lengths determined for E. coli DMSO reductase are very similar to those determined for the well-characterized Rhodobacter sphaeroides DMSO reductase, suggesting similar active site structures for the two enzymes. Furthermore, our results suggest that the form found in vivo is the monooxobis(molybdopterin) species.

Mechanism of Assembly of the Bis(Molybdopterin Guanine Dinucleotide)Molybdenum Cofactor in Rhodobacter sphaeroidesDimethyl Sulfoxide Reductase

A fully defined in vitro system has been developed for studying the mechanism of assembly of the bis(molybdopterin guanine dinucleotide)molybdenum cofactor in Rhodobacter sphaeroides dimethyl sulfoxide reductase (DMSOR). R. sphaeroides DMSOR expressed in a mobA(-) Escherichia coli strain lacks molybdopterin and molybdenum but contains a full complement of guanine in the form of GMP and GDP. Escherichia coli MobA, molybdopterin-Mo, GTP, and MgCl(2) are required and sufficient for the in vitro activation of purified DMSOR expressed in the absence of MobA. High levels of MobA inhibit the in vitro activation. A chaperone is not required for the in vitro activation process. The reconstituted DMSOR can exhibit up to 73% of the activity observed in recombinant DMSOR purified from a wild-type strain. The use of radiolabeled GTP has demonstrated incorporation of the guanine moiety from the GTP into the activated DMSOR. No role was observed for E. coli MobB in the in vitro activation of apo-DMSOR. This work also represents the first time that the MobA-mediated conversion of molybdopterin to molybdopterin guanine dinucleotide has been demonstrated directly without using the activation of a molybdoenzyme as an indicator for cofactor formation.

The 1.3 Å Crystal Structure ofRhodobacter sphaeroidesDimethyl Sulfoxide Reductase Reveals Two Distinct Molybdenum Coordination Environments

During the past four years, a substantial amount of structural information has been accumulated on the molybdoenzyme dimethyl sulfoxide (DMSO) reductase from purple bacteria. This enzyme contains a mononuclear Mo coordinated by two molybdopterin guanine dinucleotides as its single cofactor. Crystallographic studies on the enzyme from Rhodobacter sphaeroides and Rhodobacter capsulatus revealed substantial differences in the Mo coordination environment in the oxidized Mo(VI) state, despite a close structural similarity in the overall fold of the protein. The crystal structure of DMSO reductase from R. sphaeroides identified a Mo environment with a mono-oxo ligation and an asymmetric coordination by the two molybdopterins, with three short and one very long Mo−S bond. In contrast, two independent crystallographic studies of the enzyme from R. capsulatus revealed two additional Mo coordination environments: a pentacoordinated dioxo metal ligation sphere in which one molybdopterin is completely dissociated from the Mo and a heptacoordinated environment with symmetrical metal coordination by both molybdopterins and two oxo ligands. In all three structures the side chain of a serine was a ligand to the Mo. Adding to the controversy, EXAFS studies on the R. sphaeroides enzyme suggested a hexacoordinated active site geometry, whereas the same technique indicated seven ligands for the R. capsulatus enzyme. The 1.3 Å resolution crystal structure of oxidized DMSOR from R. sphaeroides presented here reveals plasticity at the active site. The Mo is discretely disordered and exists in a hexacoordinated and a pentacoordinated ligation sphere. The hexacoordinated model reconciles the existing differences in active site coordination of R. sphaeroides DMSO reductase as studied by crystallographic and EXAFS techniques. In addition, the pentacoordinated structure closely resembles one of the reported R. capsulatus crystal structures. In retrospect, the active site geometry in the previously reported 2.2 Å crystal structure of R. sphaeroides DMSO reductase appears to represent an average of the two conformations described here. Thus, structural flexibility at the active site appears to give rise to the observed differences in the Mo coordination environment.

Structure of the Molybdenum Site of Dimethyl Sulfoxide Reductase

Molybdenum K-edge X-ray absorption and Mo(V) electron paramagentic resonance (EPR) spectroscopies have been used to probe the metal coordination in oxidized and reduced forms of both wild-type and a site-directed mutant of Rhodobacter sphaeroides dimethyl sulfoxide (DMSO) reductase. We confirm our earlier findings (George, G. N.; Hilton, J.; Rajagopalan, K. V. J. Am. Chem. Soc. 1996, 118, 1113−1117) that the molybdenum site of the oxidized Mo(VI) enzyme possesses one terminal oxygen ligand (MoO) at 1.68 Å, four thiolate ligands at 2.44 Å, and one oxygen at 1.92 Å and that the dithionite-reduced Mo(IV) enzyme possesses a desoxo species with three or four Mo−S at 2.33 Å and two different Mo−O ligands at 2.16 and 1.92 Å. Mo(V) EPR indicates the presence of one exchangeable oxygen ligand, most likely an Mo−OH, in the signal-giving species, probably originating from the MoO of the oxidized enzyme (Em8.5(IV/V) = +37 mV, Em8.5(V/VI) = +83 mV). The addition of dimethyl sulfide, in the reverse of the physiological reaction, reduces the enzyme. In this form, the enzyme contains a desoxo active site with four Mo−S at 2.36 Å and two different Mo−O ligands at 1.94 and 2.14 Å. Recombinant wild-type R. sphaeroides DMSO reductase expressed in Escherichia coli initially has a dioxo structure (two MoO at 1.72 Å and four Mo−S at 2.48 Å) but assumes the wild-type Mo(VI) structure after a cycle of reduction and reoxidation. The site-directed Ser147→Cys mutant possesses a monooxo active site in the oxidized state (MoO at 1.70 Å) with five sulfur ligands (at 2.40 Å), consistent with cysteine 147 coordination to Mo. The dithionite reduced form of the mutant possesses a desoxo site also with five Mo−S ligands (at 2.37 Å) and one Mo−O at 2.12 Å. The variant has substantially different Mo(V) EPR and electrochemistry (Em8.5(IV/V) = −43 mV, Em8.5(V/VI) = +106 mV). The active-site structure and catalytic mechanism of DMSO reductase are discussed in the light of these results.

Synthesis, Structures, and Reactivity of Bis(dithiolene)molybdenum(IV,VI) Complexes Related to the Active Sites of Molybdoenzymes

The existence of a universal pterin dithiolene cofactor ligand for the molybdenum and tungsten oxotransferases supports a biological significance of the fundamental chemistry of mono- and bis(dithiolene) complexes of these elements. Members of the dimethyl sulfoxide (DMSO) reductase family of enzymes contain two pterin dithiolene ligands; at least one enzyme functions by using the minimal reaction couple MoIV + Me2SO ⇄ MoVIO + Me2S. Accordingly, the synthesis, structures, and reactivity of bis(dithiolene)Mo(IV,VI) complexes of benzene-1,2-dithiolate and related ligands have been investigated. A convenient synthesis of square pyramidal [MoIVO(S2C2R2)2]2- complexes is reported. Compounds of the type [Mo(S2C2R2)2(R‘NC)2], including [Mo(bdt)2(MeNC)2] (13), were prepared by reacting Na2(S2C2R2) and R‘NC with [MoCl4(MeCN)2] and were shown to be identical (R‘ = Me) to byproducts in the synthesis of [MoIVO(S2C2R2)2]2-. In one such reaction, the Fischer carbene complex [Mo(Me4bdt)2(MeNC)(CMe4bdt)] (16) was isolated. Silylation of [MoIVO(bdt)2]2- affords [MoIV(bdt)2(OSiButPh2)]- (8); an analogous reaction of [MoO2(bdt)2]2- yields [MoVIO(bdt)2(OSiButPh2)]- (11). The structures of square pyramidal 8 and cis-octahedral 11 reveal them to be minimal unconstrained representations of the des-oxo [MoIV(S2pd)2(O·Ser)] and mono-oxo [MoVIO(S2pd)2(O·Ser)] sites, respectively, of Rhodobacter sphaeroides DMSO reductase. This description applies in the limit of symmetrical dithiolene coordination; silyloxide is a simulator of serinate binding. Complex 8 shows limited reactivity with sulfoxides, and 11 is unreactive toward sulfides. However, 11 is reduced to 8 by tertiary phosphines; with excess phosphine, [Mo(bdt)2(PMePh2)2] (17) was formed. This compound was also prepared independently from [MoCl4(MeCN)2] and the phosphine. The compounds 13, 16, and 17 form an isoelectronic set with idealized trigonal prismatic (C2v) stereochemistry. These results complement a parallel development of bis(dithiolene)W(IV,VI) complexes as active-site analogues of tungstoenzymes (Lorber, C.; Donahue, J. P.; Goddard, C. G.; Nordlander, E.; Holm, R. H. J. Am. Chem. Soc. 1998, 120, 8102). Certain comparisons between the properties of molybdenum and tungsten bis(dithiolenes) are offered. (bdt = benzene-1,2-dithiolate(2−), S2pd = pterin dithiolene(2−).)

X-ray Absorption Spectroscopy of Dimethyl Sulfoxide Reductase from Rhodobacter sphaeroides

X-ray absorption spectroscopy at the molybdenum K-edge has been used to probe the molybdenum coordination of Rhodobacter sphaeroides dimethyl sulfoxide reductase. The molybdenum site of the oxidized protein possesses a novel Mo(VI) mono-oxo site (MoO at 1.68 A) with additional coordination by approximately four thiolate ligands at 2.44 A and probably one oxygen or nitrogen at 1.92 A. The reduced Mo(IV) form of the enzyme is a des-oxomolybdenum with 3−4 thiolates at 2.33 A and two different Mo−O/N ligands at 2.16 A and 1.92 A. Similarly, the stable Mo(V) glycerol-inhibited species is found to be a des-oxomolybdenum with approximately four thiolate ligands at 2.40 A and (probably) two similarly coordinated oxygen or nitrogen ligands at 1.96 A.

The amino acid sequence of Rhodobacter sphaeroides dimethyl sulfoxide reductase

The complete amino acid sequence of the soluble, monomeric molybdenum-containing enzyme dimethyl sulfoxide reductase from Rhodobacter sphaeroides f sp. denitrificans has been determined using a combination of gas-phase Edman sequencing of isolated peptides and direct sequencing of PCR products generated from R. sphaeroides genomic DNA. The protein comprises 777 residues corresponding to an apoenzyme molecular weight of 84,748 Da. The amino acid sequence was rich in Ala and Gly residues which represented 21% of the protein's composition. The DNA sequence was 67% rich in G and C nucleotides. The amino acid sequence contained 10 cysteine residues which were relatively evenly distributed throughout the sequence and featured regions of sequence corresponding to the prokaryotic molybdopterin-binding signatures 2 and 3. While exhibiting limited sequence similarity to the corresponding membrane-bound molybdenum-containing subunit (DmsA) of Escherichia coli dimethyl sulfoxide reductase, the Rhodobacter sequence showed extensive sequence similarity to that of the E. coli molybdoprotein, trimethylamine N-oxide reductase (torA). Comparison with other related prokaryotic molybdenum-containing enzymes indicated the presence of two highly conserved cysteine residues (Cys-268 and Cys-616) which may function in molybdenum coordination.

Molecular Cloning and Expression of Biotin Sulfoxide Reductase from Rhodobacter sphaeroides Forma Sp. Denitrificans

Biotin sulfoxide reductase catalyzes the conversion of d-biotin d-sulfoxide (BSO) to d-biotin. Oligonucleotides directed against common sequences in Escherichia coli biotin sulfoxide reductase and in Rhodobacter sphaereides f.sp. denitrificans dimethyl sulfoxide reductase have been utilized to amplify by PCR a 651-bp fragment from R. sphaeroides total genomic DNA that showed a high degree of sequence similarity with both E. coli biotin sulfoxide reductase and R. sphaeroides dimethyl sulfoxide reductase. Screening of a genomic cosmid library, prepared from R. sphaeroides genomic DNA, with this probe resulted in the isolation of a 7-kb EcoRI- EcoRI fragment that contained the complete coding region for R. sphaereides BSO reductase which has been sequenced, The sequence data indicated a single open reading frame of 2231 nucleotides encoding a protein of 744 amino acid residues corresponding to a subunit molecular weight of 80,234 Da. The translated protein sequence contained the prokaryotic Mo-pterin signatures 2 and 3 (Mo-cofactor binding motifs) and a ATP/GTP-binding P-loop, The R. sphaeroides BSO reductase sequence showed 51% sequence similarity with the corresponding E. coli enzyme. In addition, there were only two conserved cysteines between the two BSO reductase sequences. The R. sphaeroides gene was demonstrated, by complementation, to rescue a mutant E. coli strain that was deficient in BSO reductase when grown on BSO as the sole source of biotin, When expressed from the FLAG*Shift 12c expression vector, in the presence of IPTG, the BSO reductase gene encoded a protein of approximately 80 kDa, which cross-reacted with the anti-FLAG: monoclonal antibody and exhibited BSO reductase activity by the disk microbiological assay.

Direct oxygen atom transfer in the mechanism of action of Rhodobacter sphaeroides dimethyl sulfoxide reductase

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDirect oxygen atom transfer in the mechanism of action of Rhodobacter sphaeroides dimethyl sulfoxide reductaseBrian E. Schultz, Russ Hille, and R. H. HolmCite this: J. Am. Chem. Soc. 1995, 117, 2, 827–828Publication Date (Print):January 1, 1995Publication History Published online1 May 2002Published inissue 1 January 1995https://doi.org/10.1021/ja00107a031RIGHTS & PERMISSIONSArticle Views382Altmetric-Citations73LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (332 KB) Get e-Alerts Get e-Alerts