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Effects of mutation of the conserved lysine-362 in cytochrome c oxidase from Rhodobacter sphaeroides

机译：球形红球菌细胞色素c氧化酶中赖氨酸362保守突变的影响

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摘要

We describe the effects of a mutation, K362M, of the conserved lysine in cytochrome c oxidase from Rhodobacter sphaeroides, a residue located in a putative proton channel that may convey substrate protons to the binuclear center. Spectra of the "as prepared", ferricyanide-oxidized, and dithionite-reduced forms of the mutant protein confirm that the redox centers remain intact. Ligand binding kinetics of the ferricyanide-oxidized enzyme and of the dithionite-reducible fraction are similar to those of the wild type, indicating that the K channel is not the major route for CO, cyanide, formate, or peroxide entry into the structure. Protonation of the lysine residue is not redox-linked to heme a or CuB as judged from the essentially unaltered midpoint potentials of these centers in the cyanide-ligated enzyme. A difficulty in electron transfer from heme a to the binuclear center is indicated by the slow and only partial reduction of heme a3 by dithionite or ferrocytochrome c and by the presence of some reduced heme a in the as prepared mutant enzyme and under steady-state conditions. Further characterization of the K362M enzyme in the steady state shows that up to one electron, but not two, can enter the binuclear center easily. It is this inability to form the two-electron-reduced, oxygen-reactive R state that prevents activity. A model is proposed where the K channel serves as a dielectric well of high dielectric strength and low proton conductivity, rather than as a pathway for proton entry to the binuclear center. The function of this structure would be to decrease the cost of introducing a transiently uncompensated charge into the binuclear center prior to formation of a stable, charge-compensated R-state.
机译：我们描述了从球形球形红细菌（Rhodobacter sphaeroides）的细胞色素C氧化酶中保守的赖氨酸突变K362M的影响，球形推定的质子通道中的一个残基可能将底质子传递到双核中心。 “准备好的”，铁氰化物氧化和连二亚硫酸盐还原形式的突变蛋白的光谱证实氧化还原中心保持完整。铁氰化物氧化酶和连二亚硫酸盐可还原级分的配体结合动力学与野生型相似，表明K通道不是CO，氰化物，甲酸或过氧化物进入结构的主要途径。从氰化物连接的酶中这些中心的基本未改变的中点电势判断，赖氨酸残基的质子化未与血红素a或CuB氧化还原连接。电子从血红素a转移到双核中心的困难表现为连二亚硫酸盐或铁血色素c缓慢且仅部分还原了血红素a3，并且在制备的突变酶中和稳态条件下存在一些还原的血红素a。 。稳定状态下K362M酶的进一步表征表明，最多一个电子而不是两个电子可以轻松进入双核中心。正是这种不能形成两电子还原的，氧反应的R态阻止了活性。提出了一种模型，其中K通道充当具有高介电强度和低质子传导率的介电阱，而不是用作质子进入双核中心的途径。这种结构的功能是降低在形成稳定的，经电荷补偿的R状态之前将瞬态未经补偿的电荷引入双核中心的成本。

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