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首页> 外文期刊>Biophysical Chemistry: An International Journal Devoted to the Physical Chemistry of Biological Phenomena >Relocation of an internal proton donor in cytochrome c oxidase results in an altered pK(a) and a non-integer pumping stoichiometry.
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Relocation of an internal proton donor in cytochrome c oxidase results in an altered pK(a) and a non-integer pumping stoichiometry.

机译：内部质子供体在细胞色素C氧化酶中的重定位导致pK（a）改变和非整数泵浦化学计量。

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摘要

Cytochrome c oxidase from Rhodobacter sphaeroides has two proton-input pathways leading from the protein surface towards the catalytic site, located within the membrane-spanning part of the enzyme. One of these pathways, the D-pathway, contains a highly conserved Glu residue [E(I-286)], which plays an important role in proton transfer through the pathway. In a recent study, we showed that a mutant enzyme in which E(I-286) was re-located to the opposite side of the D-pathway [EA(I-286)/IE(I-112) double mutant enzyme] was able to pump protons, although with a stoichiometry that was lower than that of the wild-type enzyme ( approximately 0.6 H(+)/e(-)) (Aagaard et al. (2000) Biochemistry 39, 15847-15850). These results showed that the residue must not necessarily be located at a specific place in the amino-acid sequence, but rather at a specific location in space. In this study, we have investigated the effect of moving E(I-286) on the kinetics of specific reaction steps of the catalytic cycle in the pH range 6-11. Our results show that during the reaction of the four-electron reduced enzyme with O(2), the rates of the two first transitions (up to formation of the 'peroxy' intermediate, P(r)) are the same for the double mutant as for the wild-type enzyme, but formation of the oxo-ferryl (F) and fully oxidized (O) states, associated with proton uptake from the bulk solution, are slowed by factors of approximately 30 and approximately 400, respectively. Thus, in spite of the dramatically reduced transition rates, the proton-pumping stoichiometry is reduced only by approximately 40%. The apparent pK(a) values in the pH-dependencies of the rates of the P(R)-->F and F-->O transitions were >3 and approximately 2 units lower than those of the corresponding transitions in the wild-type enzyme, respectively. The relation between the modified pK(a)s, the transition rates between oxygen intermediates and the pumping stoichiometry is discussed(1).
机译：球形球形红细菌的细胞色素c氧化酶有两个质子输入途径，从蛋白质表面通向催化位点，位于酶的跨膜部分。这些途径之一是D途径，其中包含高度保守的Glu残基[E（I-286）]，它在通过该途径的质子转移中起重要作用。在最近的研究中，我们显示了一种突变酶，其中E（I-286）重定位到D通路的另一侧[EA（I-286）/ IE（I-112）双突变酶]尽管其化学计量比野生型酶（约0.6 H（+）/ e（-））低（Aagaard等人（2000）Biochemistry 39，15847-15850），但它能够泵送质子。这些结果表明，残基不必一定位于氨基酸序列中的特定位置，而必须位于空间中的特定位置。在这项研究中，我们已经研究了移动E（I-286）对pH范围为6-11的催化循环特定反应步骤动力学的影响。我们的结果表明，在四电子还原酶与O（2）反应期间，两个第一个跃迁的速率（直至形成“过氧化物”中间体P（r））对于双突变体而言都是相同的对于野生型酶而言，与从大批溶液中吸收质子有关的氧代-轮渡（F）和完全氧化（O）态的形成分别减慢了约30倍和约400倍。因此，尽管过渡速率大大降低，但质子泵化学计量仅降低了约40％。 P（R）-> F和F-> O转变的速率在pH依赖性中的明显pK（a）值> 3，比野生的相应转变的速率低约2个单位。型酶。讨论了修饰的pK（a）s，氧中间体之间的跃迁速率与泵送化学计量之间的关系（1）。

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