A novel bacterial sulfite dehydrogenase that requires three c-type cytochromes for electron transfer.

c-type Cytochromes (c-Cyts), primarily as electron carriers and oxidoreductases, play a key role in energy transduction processes in virtually all living organisms. Many bacteria, such as Shewanella oneidensis, are particularly rich in c-Cyts, supporting respiratory versatility not seen in eukaryotes. Unfortunately, a large number of c-Cyts are underexplored, and their biological functions remain unknown. In this study, we identify SorCABD of S. oneidensis as a novel sulfite dehydrogenase (SDH), which catalyzes the oxidation of sulfite to sulfate. In addition to catalytic subunit SorA, this enzymatic complex includes three c-Cyt subunits, which all together carry out electron transfer. The electrons extracted from sulfite oxidation are ultimately delivered to oxygen, leading to oxygen reduction, a process relying on terminal oxidase cyt cbb3. Genomic analysis suggests that the homologs of this SDH are present in a small number of bacterial genera, Shewanella and Vibrio in particular. Because these bacteria are generally capable of reducing sulfite under anaerobic conditions, the co-existence of a sulfite oxidation system implies that they may play especially important roles in the transformation of sulfur species in natural environments.Importancec-type Cytochromes (c-Cyts) endow bacteria with high flexibility in their oxidative/respiratory systems, allowing them to extracellularly transform diverse inorganic and organic compounds for survival and growth. However, a large portion of the bacterial c-Cyts remain functionally unknown. Here, we identify three c-Cyts that work together as essential electron transfer partners for the catalytic subunit of a novel SDH in sulfite oxidation in Shewanella oneidensis. This characteristic makes S. oneidensis the first organism known to be capable of oxidizing and reducing sulfite. The findings suggest that Shewanella, along with a small number of other aquatic bacteria, would serve as a particular driving force in the biogeochemical sulfur cycle in nature.

Sulfite oxidation by the quinone-reducing molybdenum sulfite dehydrogenase SoeABC from the bacterium Aquifex aeolicus.

The microaerophilic bacterium Aquifex aeolicus is a chemolitoautotroph that uses sulfur compounds as electron sources. The model of oxidation of the energetic sulfur compounds in this bacterium predicts that sulfite would probably be a metabolic intermediate released in the cytoplasm. In this work, we purified and characterized a membrane-bound sulfite dehydrogenase, identified as an SoeABC enzyme, that was previously described as a sulfur reductase. It is a member of the DMSO-reductase family of molybdenum enzymes. This type of enzyme was identified a few years ago but never purified, and biochemical data and kinetic properties were completely lacking. An enzyme catalyzing sulfite oxidation using Nitro-blue tetrazolium as artificial electron acceptor was extracted from the membrane fraction of Aquifex aeolicus. The purified enzyme is a dimer of trimer (αßγ)2 of about 390 kDa. The KM for sulfite and kcat values were 34 µM and 567 s-1 respectively, at pH 8.3 and 55 °C. We furthermore showed that SoeABC reduces a UQ10 analogue, the decyl-ubiquinone, as well, with a KM of 2.6 µM and a kcat of 52.9 s-1. It seems to specifically oxidize sulfite but can work in the reverse direction, reduction of sulfur or tetrathionate, using reduced methyl viologen as electron donor. The close phylogenetic relationship of Soe with sulfur and tetrathionate reductases that we established, perfectly explains this enzymatic ability, although its bidirectionality in vivo still needs to be clarified. Oxygen-consumption measurements confirmed that electrons generated by sulfite oxidation in the cytoplasm enter the respiratory chain at the level of quinones.

Structural evidence for a reaction intermediate mimic in the active site of a sulfite dehydrogenase.

By combining X-ray crystallography, electron paramagnetic resonance techniques and density functional theory-based modelling, we provide evidence for a direct coordination of the product analogue, phosphate, to the molybdenum active site of a sulfite dehydrogenase. This interaction is mimicking the still experimentally uncharacterized reaction intermediate proposed to arise during the catalytic cycle of this class of enzymes. This work opens new perspectives for further deciphering the reaction mechanism of this nearly ubiquitous class of oxidoreductases.

Significance of MccR, MccC, MccD, MccL and 8-methylmenaquinone in sulfite respiration of Wolinella succinogenes.

Reduction of sulfite to sulfide is an essential step in the biogeochemical sulfur cycle. The Epsilonproteobacterium Wolinella succinogenes uses the copper-containing octahaem cytochrome c sulfite reductase MccA to respire sulfite. MccA is encoded by the first gene of the mcc gene cluster, whose transcription is apparently induced by the two-component regulatory system MccRS. It has been proposed that the iron­sulfur protein MccC, the putative quinol dehydrogenase MccD, the copper chaperone MccL as well as menaquinone-6 (MK6) and/or 8-methylmenaquinone-6 (8-MMK6) are involved in the electron transport chain of W. succinogenes sulfite respiration. Here, non-polar W. succinogenes mutants were constructed that lacked MccC, MccD, MccL or the 8-MMK6-producing MK6 methyltransferase MqnK. Each mutant possessed a frameshift-corrected mccR gene, thus inducing mcc expression in the presence of a mixture of fumarate and sulfite as terminal electron acceptors. Under these conditions, growth by sulfite respiration of cells lacking MccA, MccC or MccD was found to be abolished. However, cells lacking MccL or 8-MMK6 still coupled formate oxidation to sulfite reduction and grew by sulfite respiration to some extent. The results indicate that MccR, MccC, MccD, MccL and 8-MMK6 are essential or significant components of W. succinogenes sulfite respiration.

The central active site arginine in sulfite oxidizing enzymes alters kinetic properties by controlling electron transfer and redox interactions.

A central conserved arginine, first identified as a clinical mutation leading to sulfite oxidase deficiency, is essential for catalytic competency of sulfite oxidizing molybdoenzymes, but the molecular basis for its effects on turnover and substrate affinity have not been fully elucidated. We have used a bacterial sulfite dehydrogenase, SorT, which lacks an internal heme group, but transfers electrons to an external, electron accepting cytochrome, SorU, to investigate the molecular functions of this arginine residue (Arg78). Assay of the SorT Mo centre catalytic competency in the absence of SorU showed that substitutions in the central arginine (R78Q, R78K and R78M mutations) only moderately altered SorT catalytic properties, except for R78M which caused significant reduction in SorT activity. The substitutions also altered the Mo-centre redox potentials (MoVI/V potential lowered by ca. 60-80mV). However, all Arg78 mutations significantly impaired the ability of SorT to transfer electrons to SorU, where activities were reduced 17 to 46-fold compared to SorTWT, precluding determination of kinetic parameters. This was accompanied by the observation of conformational changes in both the introduced Gln and Lys residues in the crystal structure of the enzymes. Taking into account data collected by others on related SOE mutations we propose that the formation and maintenance of an electron transfer complex between the Mo centre and electron accepting heme groups is the main function of the central arginine, and that the reduced turnover and increases in KMsulfite are caused by the inefficient operation of the oxidative half reaction of the catalytic cycle in enzymes carrying these mutations.

Phylogenetic and environmental diversity of DsrAB-type dissimilatory (bi)sulfite reductases.

The energy metabolism of essential microbial guilds in the biogeochemical sulfur cycle is based on a DsrAB-type dissimilatory (bi)sulfite reductase that either catalyzes the reduction of sulfite to sulfide during anaerobic respiration of sulfate, sulfite and organosulfonates, or acts in reverse during sulfur oxidation. Common use of dsrAB as a functional marker showed that dsrAB richness in many environments is dominated by novel sequence variants and collectively represents an extensive, largely uncharted sequence assemblage. Here, we established a comprehensive, manually curated dsrAB/DsrAB database and used it to categorize the known dsrAB diversity, reanalyze the evolutionary history of dsrAB and evaluate the coverage of published dsrAB-targeted primers. Based on a DsrAB consensus phylogeny, we introduce an operational classification system for environmental dsrAB sequences that integrates established taxonomic groups with operational taxonomic units (OTUs) at multiple phylogenetic levels, ranging from DsrAB enzyme families that reflect reductive or oxidative DsrAB types of bacterial or archaeal origin, superclusters, uncultured family-level lineages to species-level OTUs. Environmental dsrAB sequences constituted at least 13 stable family-level lineages without any cultivated representatives, suggesting that major taxa of sulfite/sulfate-reducing microorganisms have not yet been identified. Three of these uncultured lineages occur mainly in marine environments, while specific habitat preferences are not evident for members of the other 10 uncultured lineages. In summary, our publically available dsrAB/DsrAB database, the phylogenetic framework, the multilevel classification system and a set of recommended primers provide a necessary foundation for large-scale dsrAB ecology studies with next-generation sequencing methods.

Catalytic voltammetry of the molybdoenzyme sulfite dehydrogenase from Sinorhizobium meliloti.

Sulfite dehydrogenase from the soil bacterium Sinorhizobium meliloti (SorT) is a periplasmic, homodimeric molybdoenzyme with a molecular mass of 78 kDa. It differs from most other well studied sulfite oxidizing enzymes, as it bears no heme cofactor. SorT does not readily reduce ferrous horse heart cytochrome c which is the preferred electron acceptor for vertebrate sulfite oxidases. In the present study, ferrocene methanol (FM) (in its oxidized ferrocenium form) was utilized as an artificial electron acceptor for the catalytic SorT sulfite oxidation reaction. Cyclic voltammetry of FM was used to generate the active form of the mediator at the electrode surface. The FM-mediated catalytic sulfite oxidation by SorT was investigated by two different voltammetric methods, namely, (i) SorT freely diffusing in solution and (ii) SorT confined to a thin layer at the electrode surface by a semipermeable dialysis membrane. A single set of rate and equilibrium constants was used to simulate the catalytic voltammograms performed under different sweep rates and with various concentrations of sulfite and FM which provides new insights into the kinetics of the SorT catalytic mechanism. Further, we were able to model the role of the dialysis membrane in the kinetics of the overall catalytic system.

Intramolecular electron transfer between molybdenum and iron mimicking bacterial sulphite dehydrogenase.

Diferrocenyl/diferrocenium substituted dioxido molybdenum(VI) complexes [Fe2MoO2] 2(Fc)/[2(FC)]²âº mimic the catalytic active site including the redox subunits as well as the catalytic function of bacterial sulphite oxidases.

The effect of substrate and operational parameters on the abundance of sulphate-reducing bacteria in industrial anaerobic biogas digesters.

This study evaluated the effects of operational parameters and type of substrate on the abundance of sulphate-reducing bacteria in 25 industrial biogas digesters using qPCR targeting the functional dissimilatory sulphite reductase gene. The aim was to find clues for operational strategies minimizing the production of H2S. The results showed that the operation, considering strategies evaluated, only had scarce effect on the abundance, varying between 10(5) and 10(7) gene copies per ml. However, high ammonia levels and increasing concentration of sulphate resulted in significantly lower and higher levels of sulphate-reducing bacteria, respectively.

Metagenomic and biochemical characterizations of sulfur oxidation metabolism in uncultured large sausage-shaped bacterium in hot spring microbial mats.

So-called "sulfur-turf" microbial mats in sulfide containing hot springs (55-70°C, pH 7.3-8.3) in Japan were dominated by a large sausage-shaped bacterium (LSSB) that is closely related to the genus Sulfurihydrogenibium. Several previous reports proposed that the LSSB would be involved in sulfide oxidation in hot spring. However, the LSSB has not been isolated yet, thus there has been no clear evidence showing whether it possesses any genes and enzymes responsible for sulfide oxidation. To verify this, we investigated sulfide oxidation potential in the LSSB using a metagenomic approach and subsequent biochemical analysis. Genome fragments of the LSSB (a total of 3.7 Mb sequence including overlapping fragments) were obtained from the metagenomic fosmid library constructed from genomic DNA of the sulfur-turf mats. The sequence annotation clearly revealed that the LSSB possesses sulfur oxidation-related genes coding sulfide dehydrogenase (SD), sulfide-quinone reductase and sulfite dehydrogenase. The gene encoding SD, the key enzyme for sulfide oxidation, was successfully cloned and heterologously expressed in Escherichia coli. The purified recombinant enzyme clearly showed SD activity with optimum temperature and pH of 60°C and 8.0, respectively, which were consistent with the environmental conditions in the hot spring where the sulfur-turf thrives. Furthermore, the affinity of SD to sulfide was relatively high, which also reflected the environment where the sulfide could be continuously supplied. This is the first report showing that the LSSB harbors sulfide oxidizing metabolism adapted to the hot spring environment and can be involved in sulfide oxidation in the sulfur-turf microbial mats.

Roseobacter clade bacteria are abundant in coastal sediments and encode a novel combination of sulfur oxidation genes.

Roseobacter clade bacteria (RCB) are abundant in marine bacterioplankton worldwide and central to pelagic sulfur cycling. Very little is known about their abundance and function in marine sediments. We investigated the abundance, diversity and sulfur oxidation potential of RCB in surface sediments of two tidal flats. Here, RCB accounted for up to 9.6% of all cells and exceeded abundances commonly known for pelagic RCB by 1000-fold as revealed by fluorescence in situ hybridization (FISH). Phylogenetic analysis of 16S rRNA and sulfate thiohydrolase (SoxB) genes indicated diverse, possibly sulfur-oxidizing RCB related to sequences known from bacterioplankton and marine biofilms. To investigate the sulfur oxidation potential of RCB in sediments in more detail, we analyzed a metagenomic fragment from a RCB. This fragment encoded the reverse dissimilatory sulfite reductase (rDSR) pathway, which was not yet found in RCB, a novel type of sulfite dehydrogenase (SoeABC) and the Sox multi-enzyme complex including the SoxCD subunits. This was unexpected as soxCD and dsr genes were presumed to be mutually exclusive in sulfur-oxidizing prokaryotes. This unique gene arrangement would allow a metabolic flexibility beyond known sulfur-oxidizing pathways. We confirmed the presence of dsrA by geneFISH in closely related RCB from an enrichment culture. Our results show that RCB are an integral part of the microbial community in marine sediments, where they possibly oxidize inorganic and organic sulfur compounds in oxic and suboxic sediment layers.

The Wolinella succinogenes mcc gene cluster encodes an unconventional respiratory sulphite reduction system.

Assimilatory and dissimilatory sulphite reductions are key reactions in the biogeochemical sulphur cycle and several distinct sirohaem-containing sulphite reductases have been characterized. Here, we describe that the Epsilonproteobacterium Wolinella succinogenes is able to grow by sulphite respiration (yielding sulphide) with formate as electron donor. Sulphite is reduced by MccA, a prototypical member of an emerging new class of periplasmic cytochrome c sulphite reductases that, phylogenetically, belongs to a multihaem cytochrome c superfamily whose members play crucial roles in the global sulphur and nitrogen cycles. Within this family, MccA represents an unconventional octahaem cytochrome c containing a special haem c group that is bound via two cysteine residues arranged in a unique CX(15)CH haem c binding motif. The phenotypes of numerous W.succinogenes mutants producing MccA variants underlined the structural importance of this motif. Several open reading frames of the mcc gene cluster were individually inactivated and characterization of the corresponding mutants indicated that the predicted iron-sulphur protein MccC, the putative quinol dehydrogenase MccD (a member of the NrfD/PsrC family) as well as a peptidyl-prolyl cis-trans isomerase, MccB, are essential for sulphite respiration. MccA synthesis in W. succinogenes was found to be induced by sulphite (but not by thiosulphate or sulphide) and repressed in the presence of fumarate or nitrate. Based on the results, a sophisticated model of respiratory sulphite reduction by the Mcc system is presented.

Bacterial sulfite-oxidizing enzymes.

Enzymes belonging to the Sulfite Oxidase (SO) enzyme family are found in virtually all forms of life, and are especially abundant in prokaryotes as shown by analysis of available genome data. Despite this fact, only a limited number of bacterial SO family enzymes has been characterized in detail to date, and these appear to be involved in very different metabolic processes such as energy generation from sulfur compounds, host colonization, sulfite detoxification and organosulfonate degradation. The few characterized bacterial SO family enzymes also show an intriguing range of structural conformations, including monomeric, dimeric and heterodimeric enzymes with varying numbers and types of redox centres. Some of the bacterial enzymes even catalyze novel reactions such as dimethylsulfoxide reduction that previously had been thought not to be catalyzed by SO family enzymes. Classification of the SO family enzymes based on the structure of their Mo domain clearly shows that three distinct groups of enzymes belong to this family, and that almost all SOEs characterized to date are representatives of the same group. The widespread occurrence and obvious structural and functional plasticity of the bacterial SO family enzymes make this an exciting field for further study, in particular the unraveling of the metabolic roles of the three enzyme groups, some of which appear to be associated almost exclusively with pathogenic microorganisms.

Short circuiting a sulfite oxidising enzyme with direct electrochemistry: active site substitutions and their effect on catalysis and electron transfer.

Sulfite dehydrogenase (SDH) from Starkeya novella is a heterodimeric enzyme comprising a Mo active site and a heme c electron relay, which mediates electron transfer from the Mo cofactor to cytochrome c following sulfite oxidation. Studies on the wild type enzyme (SDH(WT)) and its variants have identified key amino acids at the active site, specifically Arg-55 and His-57. We report the Mo(VI/V), Mo(V/IV) and Fe(III/II) (heme) redox potentials of the variants SDH(R55K), SDH(R55M), SDH(R55Q) and SDH(H57A) in comparison with those of SDH(WT). For SDH(R55M), SDH(R55Q) and SDH(H57A) the heme potentials are lowered from ca. 240mV in SDH(WT) to ca. 200mV, while the heme potential in SDH(R55K) remains unchanged and the Mo redox potentials are not affected significantly in any of these variants. Protein film voltammetry reveals a pH dependence of the electrochemical catalytic half-wave potential (E(cat)) of -59mV/pH in SDH(WT) and SDH(R55K) which tracks the pH dependence of the Mo(VI/V) redox potential. By contrast, the catalytic potentials for SDH(R55M) and SDH(H57A) are pH-independent and follow the potential of the heme cofactor. These results highlight a switch in the pathway of electron exchange as a function of applied potential that is revealed by protein film voltammetry where an actuation of rate limiting intramolecular electron transfer (IET, Mo to heme) at high potential attenuates the catalytic current relative to faster, direct electron transfer (Mo to electrode) at lower potential. The same change in electron transfer pathway is linked to an unusual peak-shaped profile of the ideally sigmoidal steady state voltammogram in SDH(WT) alone, which has been associated with a potential dependent change in the orientation of the enzyme on the electrode surface. All other variants show purely sigmoidal voltammetry due to their inherently slower turnover numbers which are always lower than IET rates.

Highly sensitive and stable electrochemical sulfite biosensor incorporating a bacterial sulfite dehydrogenase.

This paper describes a highly sensitive electrochemical (voltammetric) determination of sulfite using a combination of Starkeya novella sulfite dehydrogenase (SDH), horse heart cytochrome c (cyt c), and a self-assembled monolayer of 11-mercaptoundecanol (MU) cast on a gold electrode. The biosensor was optimized in terms of pH and the ratio of cyt c/SDH. The electrocatalytic oxidation current of sulfite increased linearly from 1 to 6 microM at the enzyme-modified electrode with a correlation coefficient of 0.9995 and an apparent Michaelis constant (K(M,app)) of 43 microM. Using an amperometric method, the low detection limit for sulfite at the enzyme-modified electrode was 44 pM (signal-to-noise ratio = 3). The modified electrode retained a stable response for 3 days while losing only ca. 4% of its initial sensitivity during a 2 week storage period in 50 mM Tris buffer solution at 4 degrees C. The enzyme electrode was successfully used for the determination of sulfite in beer and white wine samples. The results of these electrochemical analyses agreed well with an independent spectrophotometric method using Ellman's reagent, but the detection limit was far superior using the electrochemical method.

Elucidating the catalytic mechanism of sulfite oxidizing enzymes using structural, spectroscopic, and kinetic analyses.

Sulfite oxidizing enzymes (SOEs) are molybdenum cofactor-dependent enzymes that are found in plants, animals, and bacteria. Sulfite oxidase (SO) is found in animals and plants, while sulfite dehydrogenase (SDH) is found in bacteria. In animals, SO catalyzes the oxidation of toxic sulfite to sulfate as the final step in the catabolism of the sulfur-containing amino acids, methionine and cysteine. In humans, sulfite oxidase deficiency is an inherited recessive disorder that produces severe neonatal neurological problems that lead to early death. Plant SO (PSO) also plays an important role in sulfite detoxification and in addition serves as an intermediate enzyme in the assimilatory reduction of sulfate. In vertebrates, the proposed catalytic mechanism of SO involves two intramolecular one-electron transfer (IET) steps from the molybdenum cofactor to the iron of the integral b-type heme. A similar mechanism is proposed for SDH, involving its molybdenum cofactor and c-type heme. However, PSO, which lacks an integral heme cofactor, uses molecular oxygen as its electron acceptor. Here we review recent results for SOEs from kinetic measurements, computational studies, electron paramagnetic resonance (EPR) spectroscopy, electrochemical measurements, and site-directed mutagenesis on active site residues of SOEs and of the flexible polypepetide tether that connects the heme and molybdenum domains of human SO. Rapid kinetic studies of PSO are also discussed.

Pulsed EPR investigations of the Mo(V) centers of the R55Q and R55M variants of sulfite dehydrogenase from Starkeya novella.

Continuous-wave and pulsed electron paramagnetic resonance (EPR) spectroscopy have been used to characterize two variants of bacterial sulfite dehydrogenase (SDH) from Starkeya novella in which the conserved active-site arginine residue (R55) is replaced by a neutral amino acid residue. Substitution by the hydrophobic methionine residue (SDH(R55M)) has essentially no effect on the pH dependence of the EPR properties of the Mo(V) center, even though the X-ray structure of this variant shows that the methionine residue is rotated away from the Mo center and a sulfate anion is present in the active-site pocket (Bailey et al. in J Biol Chem 284:2053-2063, 2009). For SDH(R55M) only the high-pH form is observed, and samples prepared in H(2)(17)O-enriched buffer show essentially the same (17)O hyperfine interaction and nuclear quadrupole interaction parameters as SDH(WT) enzyme. However, the pH dependence of the EPR spectra of SDH(R55Q), in which the positively charged arginine is replaced by the neutral hydrophilic glutamine, differs significantly from that of SDH(WT). For SDH(R55Q) the blocked form with bound sulfate is generated at low pH, as verified by (33)S couplings observed upon reduction with (33)S-labeled sulfite. This observation of bound sulfate for SDH(R55Q) supports our previous hypothesis that sulfite-oxidizing enzymes can exhibit multiple pathways for electron transfer and product release (Emesh et al. in Biochemistry 48:2156-2163, 2009). At pH > or = 8 the high-pH form dominates for SDH(R55Q).

Exchangeable oxygens in the vicinity of the molybdenum center of the high-pH form of sulfite oxidase and sulfite dehydrogenase.

The electron spin echo envelope modulation (ESEEM) investigation of the high-pH (hpH) form of sulfite oxidase (SO) and sulfite dehydrogenase (SDH) prepared in buffer enriched with H(2)(17)O reveals the presence of three types of exchangeable oxygen atoms at the molybdenum center. Two of these oxygen atoms belong to the equatorial OH ligand and the axial oxo ligand, and are characterized by (17)O hyperfine interaction (hfi) constants of about 37 MHz and 6 MHz, respectively. The third oxygen has an isotropic hfi constant of 3-4 MHz and likely belongs to a hydroxyl moiety hydrogen-bonded to the equatorial OH ligand. This exchangeable oxygen atom is not observed in the ESEEM spectra of the Y236F mutant of SDH, where the active site tyrosine has been replaced by phenylalanine.

Molecular basis for enzymatic sulfite oxidation: how three conserved active site residues shape enzyme activity.

Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDH R55M and SDHH57A are relatively insensitive to pH (approximately 60 and 200 s(-1), respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

Direct catalytic electrochemistry of sulfite dehydrogenase: mechanistic insights and contrasts with related Mo enzymes.

Under hydrodynamic electrochemical conditions with slow cyclic voltammetry sweep rates we have been able to probe catalytic events at the molybdenum active site of sulfite dehydrogenase (SDH) from Starkeya novella adsorbed on an edge plane graphite electrode within a polylysine film. The electrochemically driven catalytic behaviour of SDH mirrors that seen in solution assays suggesting that the adsorbed enzyme retains its native activity. However, at high sulfite concentrations, the voltammetric waveform transforms from the expected sigmoidal profile to a peak-shaped response, similar to that reported for the molybdenum enzymes DMSO reductase and nitrate reductase (NarGHI and NapAB) where a redox reaction at the active site has been associated with a switch to lower activity at high overpotentials. This is the first time a similar phenomenon has been observed in a Mo-containing oxidase/dehydrogenase, which raises a number of interesting mechanistic problems. The potential at which the activity of SDH becomes attenuated only emerges at saturating substrate conditions and occurs at a potential (ca. + 320mV vs NHE) well removed from any known redox couple in the enzyme. These results cannot be explained by the same mechanism adopted for DMSO reductase and nitrate reductase catalysis.