The nature of the phosphate complex of sulphite oxidase from electron-paramagnetic-resonance studies (original) (raw)
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The Biochemical journal, 1980
Studies of the effect of substitution with 17O on the e.p.r. spectra at 9 and 35 GHz of Mo(V) in the phosphate complex of sulphite oxidase are reported. Substitution of 17O-enriched water for normal water, for samples of the enzymes reduced by sulphite in the presence of normal phosphate, produced no detectable effect on the e.p.r. signal. If phosphate substituted with 17O was used, coupling due to 17O, producing large anisotropic splittings in the spectrum, was clearly detectable. It is concluded that phosphate is co-ordinated directly to molybdenum in the active site of the enzyme, in an equatorial type of ligand position. An oxygen ligand must be displaced from the molybdenum in the process of binding the phosphate. Implications concerning the mechanism of the enzyme reactions are discussed.
The Biochemical journal, 1982
Reduction of sulphite oxidase by sulphite at low pH values in Mes (4-morpholine-ethanesulphonic acid) buffer gives rise to a new molybdenum(V) electron-paramagnetic-resonance spectrum different from that obtained by photoreduction of the enzyme in the same medium. The spectrum is attributed to a sulphite complex of the enzyme, showing g-values of about 2.000, 1.972 and 1.963. The complex is analogous to that with the inhibitor phosphate in that it gives rise to no observable hyperfine coupling of Mo(V) to exchangeable protons.
Biochemistry Usa, 1989
The active site of sulfite oxidase has been investigated by X-ray absorption spectroscopy at the molybdenum K-edge at 4 K. We have investigated all three accessible molybdenum oxidation states, Mo(IV), Mo(V), and Mo(VI), allowing comparison with the Mo(V) electron paramagnetic resonance data for the first time. Quantitative analysis of the extended X-ray absorption fine structure indicates that the Mo(V1) oxidation state possesses two terminal oxo (M d) and approximately three thiolate-like (Mo-S-) ligands and is unaffected by changes in pH and chloride concentration. The Mo(1V) and Mo(V) oxidation states, however, each have a single oxo ligand plus one Mo-0-(or Mo-N<) bond, most probably Mo-OH, and two to three thiolate-like ligands. Both reduced forms appear to gain a single chloride ligand under conditions of low p H and high chloride concentration. t Exxon Research and Engineering Co.
Biochemistry, 1989
The active site of sulfite oxidase has been investigated by X-ray absorption spectroscopy at the molybdenum K-edge at 4 K. We have investigated all three accessible molybdenum oxidation states, Mo(IV), Mo(V), and Mo(VI), allowing comparison with the Mo(V) electron paramagnetic resonance data for the first time. Quantitative analysis of the extended X-ray absorption fine structure indicates that the Mo(V1) oxidation state possesses two terminal oxo ( M d ) and approximately three thiolate-like (Mo-S-) ligands and is unaffected by changes in pH and chloride concentration. The Mo(1V) and Mo(V) oxidation states, however, each have a single oxo ligand plus one Mo-0-(or Mo-N<) bond, most probably Mo-OH, and two to three thiolate-like ligands. Both reduced forms appear to gain a single chloride ligand under conditions of low p H and high chloride concentration.
Mo V Electron Paramagnetic Resonance of Sulfite Oxidase Revisited: The Low-pH Chloride Signal
Inorganic Chemistry, 2008
Valuable information on the active sites of molybdenum enzymes has been provided by Mo V electron paramagnetic resonance (EPR) spectroscopy. In recent years, multiple resonance techniques have been extensively used to examine details of the active-site structure, but basic continuous-wave (CW) EPR has not been re-evaluated in several decades. Here, we present a re-examination of the CW EPR spectroscopy of the sulfite oxidase low-pH chloride species and provide evidence for direct coordination of molybdenum by chloride.
Inorganic Chemistry, 2005
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.
A quantum-mechanical study of the reaction mechanism of sulfite oxidase
JBIC Journal of Biological Inorganic Chemistry, 2014
The oxidation of sulfite to sulfate by two different models of the active site of sulfite oxidase has been studied. Both protonated and deprotonated substrates were tested. Geometries were optimized with density functional theory (TPSS/def2-SV(P)) and energies were calculated either with hybrid functionals and large basis sets (B3LYP/ def2-TZVPD) including corrections for dispersion, solvation, and entropy, or with coupled-cluster theory (LCCSD(T0)) extrapolated toward a complete basis set. Three suggested reaction mechanisms have been compared and the results show that the lowest barriers are obtained for a mechanism where the substrate attacks a Mo-bound oxo ligand, directly forming a Mo-bound sulfate complex, which then dissociates into the products. Such a mechanism is more favorable than mechanisms involving a Mosulfite complex with the substrate coordinating either by the S or O atom. The activation energy is dominated by the Coulomb repulsion between the Mo complex and the substrate, which both have a negative charge of -1 or -2.
Journal of The American Chemical Society, 1994
The complexes, [Bu4Nl2[Mov102(mnt)2] ( l ) , [B~4N]~[Mo~~O(mnt)2] (2), and [Ph3PNPPh3] [EtdN] [MoV-OCl(mnt)z] ( 3 ) (mnt2-= 1,2-dicyanoethylenedithiolate) have been synthesized as possible models for active sites of sulfite oxidase which is proposed to contain molybdenum cofactor with dithiolene coordination around molybdenum. The structure of the [Bu$]+ salt of complex anion of 1 has been determined by X-ray crystallography. The compound crystallizes in space group P21/c, with a = 14.200(3) A, b = 19.402(4) -4, c = 18.967(3) A, / 3 = 95.48(1)', and Z = 4. [MovIO2(mnt)2]2-is a distorted octahedron with the oxo groups cis to each other and trans to the dithiolene sulfur atoms. The complexes 1-3 have been characterized by IR, UV-visible, 13C NMR, and negative ion FAB mass spectra. Complex 1 shows a quasireversible reduction and proton coupled electron transfer reaction. Complex 2 undergoes an one-electron reversible oxidation; but on the coulometric time scale it disproportionates to a tris dithiolene complex, [MoIv(mnt)3]2-and Moo3. Complex 2 in the presence of Cl-is oxidized irreversibly with the appearance of a new quasireversible couple corresponding to the electrochemical detection of [M0~OCl(mnt)2]~-/ [M0~~OCl(mnt)2]3-. The EPR parameters of 3 and [MovO(mnt)211-are reported. The 35,37C1 superhyperfine splitting of the chloro complex 3 is shown in relevance to Mo-Cl interaction in native sulfite oxidase. Complex 1 oxidizes HS03-to HS04-with the formation of 2 and without forming the biologically irrelevant fi-oxo Mo(V) dimer. This reaction follows enzymatic substrate saturation kinetics with apparent K M (Michaelis-Menten constant) = O.OlO(*O.OOl) M and k2 (kob at substrate saturation concentration and is proportional to V,,,) = 0.87(*0.04) s-1 in MeCN/H20 (2) Rajagopalan, K. V. In Molybdenum and Molybdenum Containing Enzymes; Coughlan, M., Ed.; Pergamon Press: Oxford, 1980. 0002-7863/94/1516-9061$04.50/0 terminal oxo group appears to be present in the Mo(V) and Mo(1V) stateskvbof the enzyme. The hydroxo ligand of the Mo(V) species is suggested to be cis to the terminal oxo group as shown by 1 H superhyperfine splitting.4 No oxomolybdoenzyme has yet
Inorganic Chemistry, 2009
Electron spin echo envelope modulation (ESEEM) investigations were carried out on samples of the low-pH (lpH) form of vertebrate sulfite oxidase (SO) prepared with 35 Cl-and 37 Cl-enriched buffers as well as with buffer containing the natural abundance of Cl isotopes. The isotope-related changes observed in the ESEEM spectra provide direct and unequivocal evidence that Cl − is located in close proximity to the Mo(V) center of lpH SO. The measured isotropic hyperfine interaction constant of about 4 MHz ( 35 Cl) suggests that the Cl − ion is either weakly coordinated to Mo(V) at its otherwise vacant axial position, trans to the oxo ligand, or is hydrogen-bonded to the equatorial exchangeable OH ligand. Scalar relativistic all-electron density functional theory (DFT) calculations of the hyperfine and nuclear quadrupole interaction parameters, along with steric and energetic arguments, strongly support the possibility that Cl − is hydrogen-bonded to the equatorial OH ligand rather than being directly coordinated to the Mo(V).