Kinetic and Structural Studies of Aldehyde Oxidoreductase from Desulfovibrio gigas Reveal a Dithiolene-Based Chemistry for Enzyme Activation and Inhibition by H2O2 (original) (raw)
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Journal of the …, 2009
Aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a member of the xanthine oxidase (XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family shows a distorted square pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V) EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerolinhibited enzyme, where the catalytically labile OH/OH 2 ligand is lost and both alcohols coordinate the Mo site in a η 2 fashion. The two adducts present a direct interaction between the molybdenum and one of the carbon atoms of the alcohol moiety, which constitutes the first structural evidence for such a bond in a biological system.
Modelling the reduced xanthine oxidase in active sulfo and inactive desulfo forms
Dalton Transactions, 2013
Synthesis of two complexes, [NBu n 4 ][Mo IV O(mnt)(S-Tol)(N-N)] (N-N = 2,2'-bipyridine (1a) or 1,10-phenanthroline (1b); mnt = maleonitriledithiolate; S-Tol = toluenethiol) are reported. These on treatment with H 2 S generate the corresponding [NBu n 4 ][Mo IV O(mnt)(SH)(N-N)] (2a and 2b) complexes bearing the susceptible hydrosulfide coordination. 2a (and 2b) upon chemical oxidation show EPR spectra with the appearance of a Mo(V) signal at = 1.976 (for 2a and 2b). Such EPR signal changed to another slow Mo(V) signal at = 1.949 at the expense of the initial signal. This conversion is accelerated in the presence of trace amount of moisture in the solvent. These data are similar to the Moco isolated from the xanthine oxidase (XO) as reported by Bray. 2a (and 2b) responds to one electron electrochemical oxidation and the generated pentavalent species responds to proton coupled electron transfer reaction. A predominantly metal centered HOMO is observed in 2a (and 2b) from DFT calculations. In the inhibited Moco of XO bearing a -SH moiety, the HOMO shows considerably less electron density residing on the Mo d xy orbital and maximum electron density is distributed to the phospho-ester group rendering the Mo(IV) center incapable of participating in the electron transfer process manifesting inhibition. † Electronic supplementary information (ESI) available: Details of X-ray crystallographic parameters, UV-Vis, FTIR spectra, DFT calculations and relevant references. CCDC reference numbers 890843, 890844, 781139 and 781140. For ESI and crystallographic data in CIF or other electronic format see ; Tel: +91-3326686464 ‡ Several non-dithiolene ligands have been employed to model the active site of molybdoenzymes. Our discussion is restricted to an analogue system with the presence of a mandatory dithiolene chelation around Mo.
Journal of Molecular Biology, 2000
The aldehyde oxidoreductase (MOD) isolated from the sulfate reducer Desulfovibrio desulfuricans (ATCC 27774) is a member of the xanthine oxidase family of molybdenum-containing enzymes. It has substrate speci-®city similar to that of the homologous enzyme from Desulfovibrio gigas (MOP) and the primary sequences from both enzymes show 68 % identity. The enzyme was crystallized in space group P6 1 22, with unit cell dimensions of a b 156.4 A Ê and c 177.1 A Ê , and diffraction data were obtained to beyond 2.8 A Ê . The crystal structure was solved by Patterson search techniques using the coordinates of the D. gigas enzyme. The overall fold of the D. desulfuricans enzyme is very similar to MOP and the few differences are mapped to exposed regions of the molecule. This is re¯ected in the electrostatic potential surfaces of both homologous enzymes, one exception being the surface potential in a region identi®able as the putative docking site of the physiological electron acceptor. Other essential features of the MOP structure, such as residues of the active-site cavity, are basically conserved in MOD. Two mutations are located in the pocket bearing a chain of catalytically relevant water molecules.
Journal of Molecular Biology, 2000
The aldehyde oxidoreductase (MOD) isolated from the sulfate reducer Desulfovibrio desulfuricans (ATCC 27774) is a member of the xanthine oxidase family of molybdenum-containing enzymes. It has substrate speci-®city similar to that of the homologous enzyme from Desulfovibrio gigas (MOP) and the primary sequences from both enzymes show 68 % identity. The enzyme was crystallized in space group P6 1 22, with unit cell dimensions of a b 156.4 A Ê and c 177.1 A Ê , and diffraction data were obtained to beyond 2.8 A Ê . The crystal structure was solved by Patterson search techniques using the coordinates of the D. gigas enzyme. The overall fold of the D. desulfuricans enzyme is very similar to MOP and the few differences are mapped to exposed regions of the molecule. This is re¯ected in the electrostatic potential surfaces of both homologous enzymes, one exception being the surface potential in a region identi®able as the putative docking site of the physiological electron acceptor. Other essential features of the MOP structure, such as residues of the active-site cavity, are basically conserved in MOD. Two mutations are located in the pocket bearing a chain of catalytically relevant water molecules.
Journal of protein chemistry, 1992
The reactivities with an excess of 5-5'-dithiobis (2-nitrobenzoic) acid (DTNB) of sulphydryl residues present in xanthine oxidase and aldehyde oxidase were studied and compared. The results show that two classes of sulphydryl groups with quite different reactivities exist in both enzymes either native or denatured. Some of the available sulphydryl residues thus react instantaneously with the DTNB; whereas the others react very slowly following pseudo-firstorder kinetics. The number of sulphydryl residues of each class and the rate constant of slowly reacting groups are, respectively, 1.7 and 0.8 in native xanthine oxidase and 1.6 ;and t.7 in native aldehyde oxidase. In denatured enzymes, the number of fast-and slow-reacting sulphydryl residues obtained are, respectively, 13.9 and 7.9 in xanthine oxidase and 5.7 and 5.4 in aldehyde oxidase. Analogously, the rate constant for the slowly reacting groups is similar for the two native enzymes, but in denatured aldehyde oxidase it is double that of denatured xanthine oxidase.
Biochemistry, 2001
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, 1eoxidation 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 Oγ 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.
J Mol Biol, 2000
DMSO reductase (DMSOR) from Rhodobacter capsulatus, well-characterised as a molybdoenzyme, will bind tungsten. Protein crystallography has shown that tungsten in W-DMSOR is ligated by the dithiolene group of the two pyranopterins, the oxygen atom of Ser147 plus another oxygen atom, and is located in a very similar site to that of molybdenum in Mo-DMSOR. These conclusions are consistent with W LIII-edge X-ray absorption, EPR and UV/visible spectroscopic data. W-DMSOR is significantly more active than Mo-DMSOR in catalysing the reduction of DMSO but, in contrast to the latter, shows no significant ability to catalyse the oxidation of DMS.