Journal
BIOCHEMISTRY
Volume 43, Issue 6, Pages 1667-1674Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi0357154
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- NIGMS NIH HHS [GM 59953] Funding Source: Medline
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Arsenite oxidase from Alcaligenes faecalis, an unusual molybdoenzyme that does not exhibit a Mo(V) EPR signal during oxidative-reductive titrations, has been investigated by protein film voltammetry. A film of the enzyme on a pyrolytic graphite edge electrode produces a sharp two-electron signal associated with reversible reduction of the oxidized Mo(VI) molybdenum center to Mo(IV). That reduction or oxidation of the active site occurs without accumulation of Mo(V) is consistent with the failure to observe a Mo(V) EPR signal for the enzyme under a variety of conditions and is indicative of an obligate two-electron center. The reduction potential for the molybdenum center, 292 mV (vs SHE) at pH 5.9 and 0degreesC, exhibits a linear pH dependence for pH 5 - 10, consistent with a two-electron reduction strongly coupled to the uptake of two protons without a pK in this range. This suggests that the oxidized enzyme is best characterized as having an L2MoO2 rather than L2MoO(OH) center in the oxidized state and that arsenite oxidase uses a spectator oxo effect to facilitate the oxo transfer reaction. The onset of the catalytic wave observed in the presence of substrate correlates well with the Mo(VI/IV) potential, consistent with catalytic electron transport that is limited only by turnover at the active site. The one-electron peaks for the iron-sulfur centers are difficult to observe by protein film voltammetry, but spectrophotometric titrations have been carried out to measure their reduction potentials: at pH 6.0 and 20degreesC, that of the [3Fe-4S] center is similar to260 mV and that of the Rieske center is similar to130 mV.
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