Proton-transport mechanisms in cytochrome c oxidase revealed by studies of kinetic isotope effects.
Proton-transport mechanisms in cytochrome c oxidase revealed by studies of kinetic isotope effects.
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DOI：
10.1016/j.bbabio.2011.03.012
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发表时间：
2011-09
影响因子：
4.3
通讯作者：
Brzezinski, Peter
中科院分区：
文献类型：
--
作者：
Johansson, Ann-Louise;Chakrabarty, Suman;Berthold, Catrine L.;Hogbom, Martin;Warshel, Arieh;Brzezinski, Peter
细胞色素c氧化酶（CytcO）是一种膜结合酶，它催化双氧分子还原为水，并利用该反应释放的大部分自由能将质子泵过膜。在球形红细菌aa3细胞色素c氧化酶中，所有被泵过膜的质子以及用于氧气还原的一半质子都通过一条特定的蛋白质内质子通道进行传递，该通道含有一个高度保守的Glu286残基。为了在分子水平上理解细胞色素c氧化酶的功能，需要解决的关键问题分别与质子从Glu286转移到“泵位点”和催化位点的时间有关。在此，我们研究了野生型细胞色素c氧化酶以及两种结构变异体的细胞色素c氧化酶中分子内质子转移反应的氢/氘动力学同位素效应的温度依赖性，其中一种变异体从溶液中摄取质子延迟，另一种变异体质子泵出与氧气还原解偶联。针对与跨膜质子泵相关的两个特定反应步骤对这些过程进行了研究，一个步骤仅涉及质子转移（过氧 - 高铁，P→F，转变），另一个步骤中相同的质子转移顺序也与电子转移到催化位点相关（高铁 - 氧化态，F→O，转变）。在理论框架内对这些反应进行分析表明，较简单的P→F反应受质子从Glu286转移到催化位点的速率限制。当相同的质子转移事件也与电子转移到催化位点相关（F→O）时，质子转移反应受蛋白质结构变化的调控，这可能确保了在存在跨膜电化学梯度的情况下质子泵的化学计量比也能得以维持。
Cytochrome c oxidase (CytcO) is a membrane-bound enzyme, which catalyzes the reduction of di-oxygen to water and uses a major part of the free energy released in this reaction to pump protons across the membrane. In the Rhodobacter sphaeroides aa3 CytcO all protons that are pumped across the membrane, as well as one half of the protons that are used for O2 reduction, are transferred through one specific intraprotein proton pathway, which holds a highly conserved Glu286 residue. Key questions that need to be addressed in order to understand the function of CytcO at a molecular level are related to the timing of proton transfers from Glu286 to a “pump site” and the catalytic site, respectively. Here, we have investigated the temperature dependencies of the H/D kinetic-isotope effects of intramolecular proton-transfer reactions in the wild-type CytcO as well as in two structural CytcO variants, one in which proton uptake from solution is delayed and one in which proton pumping is uncoupled from O2 reduction. These processes were studied for two specific reaction steps linked to transmembrane proton pumping, one that involves only proton transfer (peroxy–ferryl, P→F, transition) and one in which the same sequence of proton transfers is also linked to electron transfer to the catalytic site (ferryl–oxidized, F→O, transition). An analysis of these reactions in the framework of theory indicates that that the simpler, P→F reaction is rate-limited by proton transfer from Glu286 to the catalytic site. When the same proton-transfer events are also linked to electron transfer to the catalytic site (F→O), the proton-transfer reactions are gated by a protein structural change, which presumably ensures that the proton-pumping stoichiometry is maintained also in the presence of a transmembrane electrochemical gradient.
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影响因子：
3
作者：
Brzezinski, P;Ädelroth, P
通讯作者：
Ädelroth, P
影响因子：
4.3
作者：
Ädelroth, P;Ek, M;Brzezinski, P
通讯作者：
Brzezinski, P
影响因子：
15
作者：
Billeter, SR;Webb, SP;Hammes-Schiffer, S
通讯作者：
Hammes-Schiffer, S
DOI：
10.1073/pnas.0802512105
发表时间：
2008-08-05
影响因子：
11.1
作者：
Gorbikova, Elena A.;Belevich, Ilya;Verkhovsky, Michael I.
通讯作者：
Verkhovsky, Michael I.
影响因子：
4.3
作者：
Ådelroth, P;Karpefors, M;Brzezinski, P
通讯作者：
Brzezinski, P