We describe the effects of a mutation, K362M, of the conserved lysine in cytochrome c oxidase from Rhodobacter sphaeroides, a residue located in a putative proton channel that may convey substrate protons to the binuclear center. Spectra of the "as prepared", ferricyanide-oxidized, and dithionite-reduced forms of the mutant protein confirm that the redox centers remain intact. Ligand binding kinetics of the ferricyanide-oxidized enzyme and of the dithionite-reducible fraction are similar to those of the wild type, indicating that the K channel is not the major route for CO, cyanide, formate, or peroxide entry into the structure. Protonation of the lysine residue is not redox-linked to heme a or CuB as judged from the essentially unaltered midpoint potentials of these centers in the cyanide-ligated enzyme. A difficulty in electron transfer from heme a to the binuclear center is indicated by the slow and only partial reduction of heme a3 by dithionite or ferrocytochrome c and by the presence of some reduced heme a in the as prepared mutant enzyme and under steady-state conditions. Further characterization of the K362M enzyme in the steady state shows that up to one electron, but not two, can enter the binuclear center easily. It is this inability to form the two-electron-reduced, oxygen-reactive R state that prevents activity. A model is proposed where the K channel serves as a dielectric well of high dielectric strength and low proton conductivity, rather than as a pathway for proton entry to the binuclear center. The function of this structure would be to decrease the cost of introducing a transiently uncompensated charge into the binuclear center prior to formation of a stable, charge-compensated R-state.
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