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首页> 外文期刊>Biochemistry >Structural elements involved in proton translocation by cytochrome c oxidase as revealed by backbone amide hydrogen-deuterium exchange of the E286H mutant
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Structural elements involved in proton translocation by cytochrome c oxidase as revealed by backbone amide hydrogen-deuterium exchange of the E286H mutant

机译：E286H突变体的骨架酰胺氢-氘交换揭示了细胞色素c氧化酶参与质子易位的结构元素

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Cytochrome c oxidase is the terminal electron acceptor in the respiratory chains of aerobic organisms and energetically couples the reduction of oxygen to water to proton pumping across the membrane. The mechanisms of proton uptake, gating, and pumping have yet to be completely elucidated at the molecular level for these enzymes. For Rhodobacter sphaeroides CytcO (cytochrome aa(3)) it appears as though the E286 side chain of subunit I is a branching point from which protons are shuttled either to the catalytic site for O-2 reduction or to the acceptor site for pumped protons. Amide hydrogen -deuterium exchange mass spectrometry was used to investigate how mutation of this key branching residue to histidine (E286H) affects the structures and dynamics of four redox intermediate states. A functional characterization of this mutant reveals that E286H CytcO retains similar to 1% steady-state activity that is uncoupled from proton pumping and that proton transfer from H286 is significantly slowed. Backbone amide H-D exchange, kinetics indicates that specific regions of CytcO, perturbed by the E286H mutation, are likely to be involved in proton gating and in the exit pathway for pumped protons. The results indicate that redox-dependent conformational changes around E286 are essential for internal proton transfer. E286H CytcO, however, is incapable of these specific conformational changes and therefore is insensitive to the redox state of the enzyme. These data support a model where the side chain conformation of E286 controls proton translocation in CytcO through its interactions with the proton gate, which directs the flow of protons either to the active site or to the exit pathway. In the E286H mutant, the proton gate does not function properly and the exit channel is unresponsive. These results provide new insight into the structure and mechanism of proton translocation by CytcO.
机译：细胞色素c氧化酶是需氧生物呼吸链中的末端电子受体，它能将氧的还原与水能量耦合，从而将质子泵送穿过膜。这些酶的分子水平尚未完全阐明质子吸收，门控和泵送的机制。对于球形红球菌CytcO（细胞色素aa（3））而言，似乎亚基I的E286侧链是一个分支点，质子从该分支点穿梭到催化位点以还原O-2或泵送质子的受体位点。酰胺氢-氘交换质谱用于研究该关键分支残基向组氨酸的突变（E286H）如何影响四种氧化还原中间态的结构和动力学。此突变体的功能表征表明，E286H CytcO保留了与质子泵浦无关的类似于1％的稳态活动，并且质子从H286的转移显着减慢。骨干酰胺H-D交换动力学表明，受E286H突变干扰的CytcO的特定区域可能参与质子门控和泵送质子的出口途径。结果表明，E286周围依赖氧化还原的构象变化对于内部质子转移至关重要。然而，E286H CytcO无法实现这些特定的构象变化，因此对酶的氧化还原状态不敏感。这些数据支持一个模型，其中E286的侧链构象通过其与质子门的相互作用来控制CytcO中的质子转运，质子门将质子流引导到活性位点或出口路径。在E286H突变体中，质子门功能不正常，出口通道无响应。这些结果为CytcO质子易位的结构和机制提供了新的见解。

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