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.

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.

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.

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.

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.

Sulfite oxidizing enzymes.

Sulfite oxidizing enzymes are essential mononuclear molybdenum (Mo) proteins involved in sulfur metabolism of animals, plants and bacteria. There are three such enzymes presently known: (1) sulfite oxidase (SO) in animals, (2) SO in plants, and (3) sulfite dehydrogenase (SDH) in bacteria. X-ray crystal structures of enzymes from all three sources (chicken SO, Arabidopsis thaliana SO, and Starkeya novella SDH) show nearly identical square pyramidal coordination around the Mo atom, even though the overall structures of the proteins and the presence of additional cofactors vary. This structural information provides a molecular basis for studying the role of specific amino acids in catalysis. Animal SO catalyzes the final step in the degradation of sulfur-containing amino acids and is critical in detoxifying excess sulfite. Human SO deficiency is a fatal genetic disorder that leads to early death, and impaired SO activity is implicated in sulfite neurotoxicity. Animal SO and bacterial SDH contain both Mo and heme domains, whereas plant SO only has the Mo domain. Intraprotein electron transfer (IET) between the Mo and Fe centers in animal SO and bacterial SDH is a key step in the catalysis, which can be studied by laser flash photolysis in the presence of deazariboflavin. IET studies on animal SO and bacterial SDH clearly demonstrate the similarities and differences between these two types of sulfite oxidizing enzymes. Conformational change is involved in the IET of animal SO, in which electrostatic interactions may play a major role in guiding the docking of the heme domain to the Mo domain prior to electron transfer. In contrast, IET measurements for SDH demonstrate that IET occurs directly through the protein medium, which is distinctly different from that in animal SO. Point mutations in human SO can result in significantly impaired IET or no IET, thus rationalizing their fatal effects. The recent developments in our understanding of sulfite oxidizing enzyme mechanisms that are driven by a combination of molecular biology, rapid kinetics, pulsed electron paramagnetic resonance (EPR), and computational techniques are the subject of this review.

Kinetic and structural evidence for the importance of Tyr236 for the integrity of the Mo active site in a bacterial sulfite dehydrogenase.

The sulfite dehydrogenase from Starkeya novella is the only known sulfite-oxidizing enzyme that forms a permanent heterodimeric complex between a molybdenum and a heme c-containing subunit and can be crystallized in an electron transfer competent conformation. Tyr236 is a highly conserved active site residue in sulfite oxidoreductases and has been shown to interact with a nearby arginine and a molybdenum-oxo ligand that is involved in catalysis. We have created a Tyr236 to Phe substitution in the SorAB sulfite dehydrogenase. The purified SDH(Y236F) protein has been characterized in terms of activity, structure, intramolecular electron transfer, and EPR properties. The substituted protein exhibited reduced turnover rates and substrate affinity as well as an altered reactivity toward molecular oxygen as an electron acceptor. Following reduction by sulfite and unlike SDH(WT), the substituted enzyme was reoxidized quickly in the presence of molecular oxygen, a process reminiscent of the reactions of the sulfite oxidases. SDH(Y236F) also exhibited the pH-dependent CW-EPR signals that are typically observed in vertebrate sulfite oxidases, allowing a direct link of CW-EPR properties to changes caused by a single-amino acid substitution. No quantifiable electron transfer was seen in laser flash photolysis experiments with SDH(Y236F). The crystal structure of SDH(Y236F) clearly shows that as a result of the substitution the hydrogen bonding network surrounding the active site is disturbed, resulting in an increased mobility of the nearby arginine. These disruptions underline the importance of Tyr236 for the integrity of the substrate binding site and the optimal alignment of Arg55, which appears to be necessary for efficient electron transfer.

Structure of the active site of sulfite dehydrogenase from Starkeya novella.

In this paper, we report the results of molybdenum K-edge X-ray absorption studies performed on the oxidized and reduced active sites of the sulfite dehydrogenase from Starkeya novella. Our results provide the first direct structural information on the active site of the oxidized form of this enzyme and confirm the conclusions derived from protein crystallography that the molybdenum coordination is analogous to that of the sulfite oxidases. The molybdenum atom of the oxidized enzyme is bound by two Mo=O ligands at 1.73 A and three thiolate Mo-S ligands at 2.42 A, whereas the reduced enzyme has one oxo at 1.74 A, one long oxygen at 2.19 A (characteristic of Mo-OH2), and three Mo-S ligands at 2.40 A.

Pulsed EPR studies of a bacterial sulfite-oxidizing enzyme with pH-invariant hyperfine interactions from exchangeable protons.

Variable-frequency pulsed electron paramagnetic resonance studies of the molybdenum(V) center of sulfite dehydrogenase (SDH) clearly show couplings from nearby exchangeable protons that are assigned to a Mo(V)OH(n) group. The hyperfine parameters for these exchangeable protons of SDH are the same at both low and high pH and similar to those for the high-pH forms of sulfite oxidases (SOs) from eukaryotes. The SDH proton parameters are distinctly different from the low-pH forms of chicken and human SO.