Mutants of the Cu-A site in cytochrome c oxidase of Rhodobacter sphaeroides: II. Rapid kinetic analysis of electron transfer

by Wang, Kefei; Geren, Lois; Zhen, Yuejun; Ma, Ling; Ferguson-Miller, Shelagh; Durham, Bill; Millett, Francis

The function of the binuclear CUA center in cytochrome c oxidase (CcO) was studied using two Rhodobacter sphaeroides CcO mutants involving direct ligands of the CUA center, H260N and M263L. The rapid electron-transfer kinetics of the mutants were studied by flash photolysis of a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine-55. The rate constant for intracomplex electron transfer from heme c to CUA was decreased from 40 000 s(-1) for wild-type CcO to 16 000 s(-1) and 11 000 s(-1) for the M263L and H260N mutants, respectively. The rate constant for electron transfer from CUA to heme a was decreased from 90 000 s(-1) for wild-type CcO to 4000 s(-1) for the M263L mutant and only 45 s(-1) for the H260N mutant. The rate constant for the reverse reaction, heme a to CUA, was calculated to be 66 000 s(-1) for M263L and 180 s(-1) for H260N, compared to 17 000 s(-1) for wild-type CcO. It was estimated that the redox potential of CUA was increased by 120 mV for the M263L mutant and 90 mV for the H260N mutant, relative to the potential of heme a. Neither mutation significantly affected the binding interaction with cytochrome c. These results indicate that His-260, but not Met-263, plays a significant role in electron transfer between CUA and heme a.

Start Page
1520-4995; 0006-2960