In this study we have combined the use of site-directed mutants with time-resolved optical absorption spectroscopy to investigate the role of the protonatable subunit-I residues lysine-362 (K(I362)) and threonine-359 (T(I- 359)) in cytochrome c oxidase from Rhodobacter sphaeroides in electron and proton transfer. These residues have been proposed to be part of a proton- transfer pathway in cytochrome oxidases from Paracoccus denitrificans and bovine heart. Mutation of K(I-362) and T(I359) to methionine and alanine, respectively, results in reduction of the overall turnover activities to <2% and ~ 35%, respectively, of those in the wild-type enzyme. The results show that in the absence of dioxygen, electron transfer between hemes a3 and a with a time constant of ~ 3 μs, not coupled to protonation reactions, is not affected in the mutant enzymes. However, the slower electron transfer between hemes a3 and a, coupled to proton release with a time constant of ~ 3 ms (at pH 9.0) is impaired in the KM(I-362) and TA(I-359) mutant enzymes. This is consistent with the slow reduction rate of heme a3 in the oxidized KM(I-362) enzyme because in the wild-type enzyme reduction of heme a3 is coupled to proton uptake. On the other hand, when reacting with O2, both the wild-type and mutant fully reduced enzymes become oxidized in ~ 5 ms, and proton uptake on this time scale is not affected. Hence, the results indicate that the KM(I-362) mutant enzyme is inactive because the proton-transfer pathway through K(I-362) and T(I-359) is involved in proton uptake during reduction of the oxidized binuclear center. Proton uptake during oxidation of the fully reduced enzyme takes place through a different pathway [through E(I-286) (Adelroth, P., et al. (1997) Biochemistry 36, 13824-13829)].
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