pH Dependence of the Reduction of Dioxygen to Water by Cytochrome c Oxidase. 1. The PR State Is a pH-Dependent Mixture of Three Intermediates, A, P
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- 作者：Ned Van Eps ; Istvan Szundi ; and Ó ; lö ; f Einarsdó ; ttir
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：May 6, 2003
- 年：2003
- 卷：42
- 期：17
- 页码：5065 - 5073
- 全文大小：209K
- 年卷期：v.42,no.17(May 6, 2003)
- ISSN：1520-4995
文摘
Recent studies on cytochrome oxidase have indicated that the putative "peroxy" intermediatein the catalytic cycle (PR) is a mixture of intermediates, including P and F [Sucheta, A., et al. (1998)Biochemistry 37, 17905-17914], and the bench-made P and F forms appear to have the same redox state(Fea34+=O2-), but a different protonation state [Fabian, M., and Palmer, G. (2001) Biochemistry 40, 1867-1874]. To explore the possibility that the putative PR state is a pH-dependent mixture of intermediates,we investigated the reduction of dioxygen to water by the fully reduced cytochrome oxidase at pH 6.2,7.5, and 8.5 in the visible and Soret regions (350-800 nm) using the CO flow-flash technique. Singularvalue decomposition and global exponential fitting of the time-resolved absorption difference spectraresolved five apparent lifetimes. The fastest three (1.5, 13, and 34 
s) were independent of pH, while thetwo slowest rates (80-240 s and 1.1-2.4 ms) decreased by a factor of 2-3 as the pH increased. Whenthe time-resolved spectra were analyzed using a unidirectional sequential model, the spectra of the reducedenzyme and the dioxygen-bound intermediate, compound A, were found to be pH-independent. However,the putative PR intermediate was best represented by a pH-dependent mixture of compound A, P, and F.The ferryl form was favored at low pH. The subsequent intermediate is a ferryl with a pH-dependentelectron transfer equilibrium between heme a and CuA, the reduced heme a being favored at low pH.These results suggest a pH-dependent reaction mechanism of the reduction of dioxygen to water by thefully reduced enzyme that is more complex than previously proposed.
s) were independent of pH, while thetwo slowest rates (80-240 s and 1.1-2.4 ms) decreased by a factor of 2-3 as the pH increased. Whenthe time-resolved spectra were analyzed using a unidirectional sequential model, the spectra of the reducedenzyme and the dioxygen-bound intermediate, compound A, were found to be pH-independent. However,the putative PR intermediate was best represented by a pH-dependent mixture of compound A, P, and F.The ferryl form was favored at low pH. The subsequent intermediate is a ferryl with a pH-dependentelectron transfer equilibrium between heme a and CuA, the reduced heme a being favored at low pH.These results suggest a pH-dependent reaction mechanism of the reduction of dioxygen to water by thefully reduced enzyme that is more complex than previously proposed.