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首页> 外文期刊>Biochemistry >Effects of mutation of the conserved glutamic acid-286 in subunit I of cytochrome c oxidase from Rhodobacter sphaeroides.
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Effects of mutation of the conserved glutamic acid-286 in subunit I of cytochrome c oxidase from Rhodobacter sphaeroides.

机译：球形球形红细菌细胞色素C氧化酶亚基I中保守性谷氨酸286突变的影响。

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摘要

We have studied the effects of mutations, E286Q and E286D, of the conserved glutamate in subunit I of cytochrome c oxidase from Rhodobacter sphaeroides with a view to evaluating the role of this residue in redox-linked proton translocation. The mutation E286D did not have any dramatic effects on enzyme properties and retained 50% of wild-type catalytic activity. For E286Q a fraction of the binuclear center was trapped in an unreactive, spectrally distinct form which is most likely due to misfolded protein, but the majority of E286Q reacted normally with formate and cyanide in the oxidized state, and with carbon monoxide and cyanide in the dithionite-reduced form. The mutation also had little effect on the pH-dependent redox properties of haem a in the reactive fraction. However, formation of the P state from oxidized enzyme with hydrogen peroxide or by aerobic incubation with carbon monoxide was inhibited. In particular, only an F-type product was obtained, at less than 25% yield, in the reaction with hydrogen peroxide. The aerobic steady state in the presence of ferrous cytochrome c was characterized by essentially fully reduced haem a and ferric haem a3, suggesting that the mutation hinders electron transfer from haem a to the binuclear center. Under these conditions or after reoxidation, on a seconds time scale, of haem a3 following anaerobiosis, there was no indication of accumulation of significant amounts of P state. We propose that the glutamate is implicated in several steps in the catalytic cycle, O --> R, P --> F, and, possibly, F --> O. The results are discussed in relation to the "glutamate trap" model for proton translocation.
机译：我们已经研究了球形球形红细菌细胞色素c氧化酶亚基I中保守谷氨酸突变E286Q和E286D的作用，以评估该残基在氧化还原相关质子转运中的作用。突变E286D对酶的性质没有任何显着影响，并保留了50％的野生型催化活性。对于E286Q，双核中心的一小部分以不活泼的，光谱上不同的形式被捕获，这很可能是由于蛋白质折叠错误造成的，但是大多数E286Q正常地与甲酸酯和氰化物处于氧化态反应，与一氧化碳和氰化物处于氧化态。连二亚硫酸盐还原的形式。该突变也对反应级分中血红素a的pH依赖性氧化还原特性影响很小。但是，抑制了由过氧化氢氧化的酶或通过与一氧化碳的有氧孵育而形成的P态。特别地，在与过氧化氢的反应中仅以小于25％的收率获得了F型产物。亚铁细胞色素c存在下的有氧稳态的特征是血红素a和铁血红素a3基本上被完全还原，这表明该突变阻碍了电子从血红素a转移到双核中心。在这些条件下或厌氧菌感染后血红素a3在几秒钟的时间内再氧化后，没有迹象表明大量P状态蓄积。我们建议谷氨酸参与催化循环的多个步骤，即O-> R，P-> F，甚至可能是F->O。讨论了与“谷氨酸陷阱”模型有关的结果质子易位。

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