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pH dependence of the reduction of dioxygen to water by cytochrome c oxidase. 1. The P(R) state is a pH-dependent mixture of three intermediates, A, P, and F.

Abstract：

:Recent studies on cytochrome oxidase have indicated that the putative "peroxy" intermediate in the catalytic cycle (P(R)) 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 (Fe(a3)(4+)=O(2-)), but a different protonation state [Fabian, M., and Palmer, G. (2001) Biochemistry 40, 1867-1874]. To explore the possibility that the putative P(R) 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. Singular value decomposition and global exponential fitting of the time-resolved absorption difference spectra resolved five apparent lifetimes. The fastest three (1.5, 13, and 34 micros) were independent of pH, while the two slowest rates (80-240 micros and 1.1-2.4 ms) decreased by a factor of 2-3 as the pH increased. When the time-resolved spectra were analyzed using a unidirectional sequential model, the spectra of the reduced enzyme and the dioxygen-bound intermediate, compound A, were found to be pH-independent. However, the putative P(R) 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-dependent electron transfer equilibrium between heme a and Cu(A), 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 the fully reduced enzyme that is more complex than previously proposed.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Van Eps N,Szundi I,Einarsdóttir O

doi

10.1021/bi020482m

subject

Has Abstract

pub_date

2003-05-06 00:00:00

pages

5065-73

issue

17

eissn

0006-2960

issn

1520-4995

journal_volume

42

pub_type

杂志文章

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