Hoke Kevin R, Cobb Nathan, Armstrong Fraser A, Hille Russ
Inorganic Chemistry Laboratory, Oxford University, South Parks Road, Oxford OX1 3QR, England.
Biochemistry. 2004 Feb 17;43(6):1667-74. doi: 10.1021/bi0357154.
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 0 degrees C, 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 L(2)MoO(2) rather than L(2)MoO(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 20 degrees C, that of the [3Fe-4S] center is approximately 260 mV and that of the Rieske center is approximately 130 mV.
粪产碱杆菌中的亚砷酸盐氧化酶是一种不同寻常的钼酶，在氧化还原滴定过程中不显示Mo(V)的电子顺磁共振信号，已通过蛋白质膜伏安法进行了研究。在热解石墨边缘电极上的酶膜产生一个尖锐的双电子信号，该信号与氧化态的Mo(VI)钼中心可逆还原为Mo(IV)有关。活性位点的还原或氧化过程中没有Mo(V)的积累，这与在各种条件下未观察到该酶的Mo(V)电子顺磁共振信号一致，表明它是一个专性双电子中心。钼中心的还原电位在pH 5.9和0℃时为292 mV（相对于标准氢电极），在pH 5 - 10范围内呈线性pH依赖性，这与双电子还原过程与两个质子的摄取紧密耦合且在此范围内没有pK值一致。这表明氧化态的酶最好被表征为具有L(2)MoO(2)而不是氧化态的L(2)MoO(OH)中心，并且亚砷酸盐氧化酶利用“旁观氧代”效应来促进氧代转移反应。在底物存在下观察到的催化波的起始点与Mo(VI/IV)电位密切相关，这与仅受活性位点周转限制的催化电子传递一致。蛋白质膜伏安法很难观察到铁硫中心的单电子峰，但已进行分光光度滴定以测量它们的还原电位：在pH 6.0和20℃时，[3Fe - 4S]中心的还原电位约为260 mV，Rieske中心的还原电位约为130 mV。
