The nature of the phosphate complex of sulphite oxidase from electron-paramagnetic-resonance studies.
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ABSTRACT:
SUBMITTER: George GN
PROVIDER: S-EPMC1135405 | biostudies-other | 1988 Nov
REPOSITORIES: biostudies-other
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Project description: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.
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Project description: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.
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Project description:A study has been made of e.p.r. signals due to Mo(V) in reduced sulphite oxidase (EC 1.8.3.1) from chicken liver. Reduction by SO3(2-), or photochemically in the presence of a deazaflavin derivative, produces spectra indistinguishable from one another. Three types of spectra from the enzyme were distingusihed and shown to correspond to single chemical species, since they could be simulated at both 9 and 35 GHz by using the same parameters. These were the low-pH form of the enzyme, with gav. 1.9805, the high-pH form, with gav. 1.9681 and a phosphate complex, with gav. 1.9741. The low-H form shows interaction with a single exchangeable proton, with A(1H)av. (hyperfine coupling constant) = 0.98 mT, probably in the form of an MoOH group. Parameters of the signals are compared with those for signals from xanthine oxidase and nitrate reductase. The signal from the phosphate complex of sulphite oxidase in unique among anion complexes of Mo-containing enzymes in showing no hyperfine coupling to protons. There is no evidence for additional weakly coupled protons or nitrogen nuclei in the sulphite oxidase signals. The possibility is considered that the enzymic mechanism involves abstraction of a proton and two electrons from HSO3- by a Mo = O group in the enzyme.
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Project description:Molybdenum(V) e.p.r. spectra from reduced forms of aldehyde oxidase were obtained and compared with those from xanthine oxidase. Inhibited and Desulpho Inhibited signals from aldehyde oxidase were fully characterized, and parameters were obtained with the help of computer simulations. These differ slightly but significantly from the corresponding parameters for the xanthine oxidase signals. Rapid type 1 and type 2 and Slow signals were obtained from aldehyde oxidase, but were not fully characterized. From the general similarities of the signals from the two enzymes, it is concluded that the ligands of molybdenum must be identical and that the overall co-ordination geometries must be closely similar in the enzymes. The striking differences in substrate specificity must relate primarily to structural differences in a part of the active centre concerned with substrate binding and not involving the catalytically important molybdenum site.
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