Factors determining electron-transfer rates in cytochrome c oxidase:: investigation of the oxygen reaction in the R-sphaeroides enzyme
Factors determining electron-transfer rates in cytochrome c oxidase:: investigation of the oxygen reaction in the R-sphaeroides enzyme
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DOI：
10.1016/s0005-2728(98)00142-x
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发表时间：
1998-10-05
影响因子：
4.3
通讯作者：
Brzezinski, P
中科院分区：
文献类型：
--
作者：
Ädelroth, P;Ek, M;Brzezinski, P
我们利用流动闪光技术研究了球形红细菌中完全还原的可溶性细胞色素c氧化酶（细胞色素aa(3)）与分子氧的单周转反应动力学，并将结果与特性明确的牛线粒体酶所获结果进行了比较。两种酶的总体反应顺序相同，但电子传递反应的程度和速率不同，这意味着在氧还原过程中氧化还原电位和/或电子与质子之间的相互作用能存在差异。与牛酶一样，球形红细菌酶在pH 7.9时显示出两个主要的质子摄取动力学阶段，速率常数分别约为5000 s⁻¹和500 s⁻¹，这与过氧态到氧还铁（Ⅳ）态以及氧还铁（Ⅳ）态到氧化态的转变同时发生。球形红细菌酶摄取的质子净数量约为1.9，这意味着在还原时，该酶必须从介质中摄取约2.1个H⁺。基于对两种酶中电子传递反应的比较，我们得出结论：第四个电子向双核中心的传递速率不仅由血红素a向双核中心的电子传递速率决定，还由Cu - A和血红素a之间的电子平衡决定。此外，与牛酶不同的是，在牛酶中，完全还原的酶被O₂氧化时的电子和质子传递速率都比总周转速率快，而在球形红细菌酶中，最慢的动力学阶段对总周转起限速作用。而且，对两种体系中反应的比较表明，在球形红细菌酶中，氧还原过程中电子在氧化还原中心之间的分布更均匀。这使得研究例如突变酶中电子传递特性的微小扰动影响成为可能，本研究为此奠定了基础。（C）1998年，爱思唯尔科学出版社。版权所有。
We have investigated the kinetics of the single-turnover reaction of fully reduced solubilised cytochrome c oxidase (cytochrome aa(3)) from Rhodobacter sphaeroides with dioxygen using the flow-flash methodology and compared the results to those obtained with the well-characterised bovine mitochondrial enzyme. The overall reaction sequence was the same in the two enzymes, but the extents and rates of the electron-transfer reactions differed, implying differences in redox potentials, and/or interaction energies between electrons and protons during oxygen reduction. As with the bovine enzyme, the R. sphaeroides enzyme displayed two major kinetic phases of proton uptake with rate constants of similar to 5000 s(-1) and similar to 500 s(-1) at pH 7.9, concomitant with the peroxy to oxoferryl and oxoferryl to oxidised states. The net number of protons taken up in the R sphaeroides enzyme was about similar to 1.9, which implies that upon reduction, the enzyme has to pick up similar to 2.1 H+ from the medium.On the basis of the comparison of electron-transfer reactions in the two enzymes, we conclude that the transfer rate of the fourth electron to the binuclear centre is not only determined by the electron-transfer rate from haem a to the binuclear centre, but also by the electron equilibrium between Cu-A and haem a. In addition, in contrast to the bovine enzyme, where the electron- and proton-transfer rates during oxidation of the fully reduced enzyme by O-2 are all faster than the overall turnover rate, in the R sphaeroides enzyme, the slowest kinetic phase was rate limiting for the overall turnover. Moreover, the comparison of the reactions in the two systems shows that in the R. sphaeroides enzyme, the electrons are more evenly distributed among the redox centres during oxygen reduction. This enables investigations of effects also of minor perturbations on, e.g., the electron-transfer characteristics in mutant enzymes, for which this study forms the basis. (C) 1998 Elsevier Science B.V. All rights reserved.