Proton transfer from glutamate 286 determines the transition rates between oxygen intermediates in cytochrome c oxidase
Proton transfer from glutamate 286 determines the transition rates between oxygen intermediates in cytochrome c oxidase
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DOI：
10.1016/s0005-2728(00)00194-8
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发表时间：
2000-08-15
影响因子：
4.3
通讯作者：
Brzezinski, P
中科院分区：
文献类型：
--
作者：
Ådelroth, P;Karpefors, M;Brzezinski, P
研究了类球红细菌细胞色素c氧化酶中过氧基（PR）→氧代铁基（F）和F →氧化态（O）的电子-质子耦合。在两种不同的突变酶中研究了这些反应的动力学：（1）艾德（I-286），其中D-途径中的关键残基之一E（I-286）被具有比谷氨酸短的侧链的天冬氨酸替代，和（2）ML（II-263），其中CUA的氧化还原电位增加了约100 mV，其在F->O跃迁期间将电子转移到双核中心的速度减慢了约200倍。在艾德（I-286）中，P-R -> F期间的质子摄取减慢了约5倍，这表明E（I-286）是PR的质子供体。此外，在突变酶中，F->O转换速率显示出约2.5的氘同位素效应，而在野生型酶中约为7。由于整个氘同位素效应被证明是与一个单一的质子转移反应，其中质子供体和受体必须相互接近（M。Karpefors，P. Angstrom delroth，P. Brzezinski，Biochemistry 39（2000）6850），艾德（I-286）中较小的氘同位素效应表明来自E（I-286）的质子转移也决定了F->O跃迁的速率。在ML（II-263）中，电子转移到双核中心的速度比通过D-途径的固有质子转移速度慢。然而，电子和质子转移到双核中心显示出类似于8的氘同位素效应，即，与野生型酶中的相同，这表明这些反应是紧密耦合的。(C)2000 Elsevier Science B. V.保留所有权利。
We have investigated the electron-proton coupling during the peroxy (PR) to oxo-ferryl (F) and F to oxidised (O) transitions in cytochrome c oxidase from Rhodobacter sphaeroides. The kinetics of these reactions were investigated in two different mutant enzymes: (1) ED(I-286), in which one of the key residues in the D-pathway, E(I-286), was replaced by an aspartate which has a shorter side chain than that of the glutamate and, (2) ML(II-263), in which the redox potential of CUA is increased by similar to 100 mV, which slows electron transfer to the binuclear centre during the F-->O transition by a factor of similar to 200. In ED(I-286) proton uptake during P-R --> F was slowed by a factor of similar to 5, which indicates that E(I-286) is the proton donor to PR. In addition, in the mutant enzyme the F-->O transition rate displayed a deuterium isotope effect of similar to 2.5 as compared with similar to 7 in the wild-type enzyme. Since the entire deuterium isotope effect was shown to be associated with a single proton-transfer reaction in which the proton donor and acceptor must approach each other (M. Karpefors, P. Angstrom delroth, P. Brzezinski, Biochemistry 39 (2000) 6850), the smaller deuterium isotope effect in ED(I-286) indicates that proton transfer from E(I-286) determines the rate also of the F-->O transition. In ML(II-263) the electron-transfer to the binuclear centre is slower than the intrinsic proton-transfer rate through the D-pathway. Nevertheless, both electron and proton transfer to the binuclear centre displayed a deuterium isotope effect of similar to 8, i.e., about the same as in the wild-type enzyme, which shows that these reactions are intimately coupled. (C) 2000 Elsevier Science B.V. All rights reserved.