水通过细胞色素氧化酶在质子运输中的复杂作用,Journal of the American Chemical Society

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水通过细胞色素氧化酶在质子运输中的复杂作用
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2010-11-17 , DOI: 10.1021/ja107244g
Hyun Ju Lee ₁ , Emelie Svahn , Jessica M J Swanson , Håkan Lepp , Gregory A Voth , Peter Brzezinski , Robert B Gennis

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细胞色素 c 氧化酶 (CytcO) 是呼吸链中的最终电子受体，催化 O(2) 还原为 H(2)O，同时将质子泵入线粒体内膜或细菌膜，以维持跨膜电化学梯度，例如，ATP 合成。在这项工作中，在初步计算研究中预测会改变质子易位和酶活性的突变通过广泛的实验和计算分析进行表征。这些突变是在来自球状红杆菌的 CytcO 中的 D 途径中引入的，D 途径是两种质子摄取途径之一。丝氨酸残基 200 和 201 与 D 通路中途的结晶解析水分子氢键结合，被更庞大的疏水残基取代（Ser200Ile、Ser200Val/Ser201Val、和 Ser200Val/Ser201Tyr) 来查询改变局部结构对酶活性以及质子摄取、释放和中间转换的影响。此外，通过阻断通路入口处的质子摄取（除上述突变外的 Asp132Asn 置换）研究了这些突变对内部质子转移的影响。尽管所有突变体 CytcO 的整体活性都降低了，但 Ser200Ile 和 Ser200Val/Ser201Val 变体都保持了泵送质子的能力。降低的活性被证明是由于在 P(R) → F 和 F → O 转变过程中氧化动力学减慢（P（R）是四电子还原反应后在催化位点形成的“过氧”中间体） CytcO 与 O(2)，F 是氧化铁中间体，O 是完全氧化的 CytcO)。此外，在 Ser200Val/ 中，P(R) → F 转变显示基本上与 pH 无关，直到 pH 12（即，Glu286 的表观 pK(a) 从 9.4 增加至少 3 pK(a) 单位） Ser201Val 突变体。突变酶中质子传输的显式模拟表明，溶剂化动力学会导致有趣的能量结果，因此提供了在静态结构或具有固定质子化状态（即缺乏显式质子传输）的系统模拟中永远不会检测到的机械见解。根据 CytcO 的质子泵送机制讨论结果。突变酶中质子传输的显式模拟表明，溶剂化动力学会导致有趣的能量结果，因此提供了在静态结构或具有固定质子化状态（即缺乏显式质子传输）的系统模拟中永远不会检测到的机械见解。根据 CytcO 的质子泵送机制讨论结果。突变酶中质子传输的显式模拟表明，溶剂化动力学会导致有趣的能量结果，因此提供在静态结构或具有固定质子化状态（即缺乏显式质子传输）的系统模拟中永远不会检测到的机械见解。根据 CytcO 的质子泵送机制讨论结果。

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Intricate Role of Water in Proton Transport through CytochromecOxidase

Cytochrome c oxidase (CytcO), the final electron acceptor in the respiratory chain, catalyzes the reduction of O(2) to H(2)O while simultaneously pumping protons across the inner mitochondrial or bacterial membrane to maintain a transmembrane electrochemical gradient that drives, for example, ATP synthesis. In this work mutations that were predicted to alter proton translocation and enzyme activity in preliminary computational studies are characterized with extensive experimental and computational analysis. The mutations were introduced in the D pathway, one of two proton-uptake pathways, in CytcO from Rhodobacter sphaeroides . Serine residues 200 and 201, which are hydrogen-bonded to crystallographically resolved water molecules halfway up the D pathway, were replaced by more bulky hydrophobic residues (Ser200Ile, Ser200Val/Ser201Val, and Ser200Val/Ser201Tyr) to query the effects of changing the local structure on enzyme activity as well as proton uptake, release, and intermediate transitions. In addition, the effects of these mutations on internal proton transfer were investigated by blocking proton uptake at the pathway entrance (Asp132Asn replacement in addition to the above-mentioned mutations). Even though the overall activities of all mutant CytcO's were lowered, both the Ser200Ile and Ser200Val/Ser201Val variants maintained the ability to pump protons. The lowered activities were shown to be due to slowed oxidation kinetics during the P(R) → F and F → O transitions (P(R) is the "peroxy" intermediate formed at the catalytic site upon reaction of the four-electron-reduced CytcO with O(2), F is the oxoferryl intermediate, and O is the fully oxidized CytcO). Furthermore, the P(R) → F transition is shown to be essentially pH independent up to pH 12 (i.e., the apparent pK(a) of Glu286 is increased from 9.4 by at least 3 pK(a) units) in the Ser200Val/Ser201Val mutant. Explicit simulations of proton transport in the mutated enzymes revealed that the solvation dynamics can cause intriguing energetic consequences and hence provide mechanistic insights that would never be detected in static structures or simulations of the system with fixed protonation states (i.e., lacking explicit proton transport). The results are discussed in terms of the proton-pumping mechanism of CytcO.

更新日期：2010-11-17

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