In the membrane-bound redox-driven proton pump cytochrome c oxidase, electron- and proton-transfer reactions must be coupled, which requires controlled modulation of the kinetic and/or thermodynamic properties of proton-transfer reactions through the membrane-spanning part of the protein. In this study we have investigated proton-transfer reactions through a pathway that is used for the transfer of both substrate and pumped protons in cytochrome c oxidase from Rhodobacter sphaeroides. Specifically, we focus on the formation of the so-called F intermediate, which is rate limited by an internal proton-transfer reaction from a possible branching point in the pathway, at a glutamic-acid residue (E(I-286)), to the binuclear center. We have also studied the reprotonation of E(I-286) from the bulk solution. Evaluation of the data in terms of a model presented in this work gives a rate of internal proton transfer from E(I-286) to the proton acceptor at the catalytic site of 1.1 x 10(4) s(-1). The apparent pK(a) of the donor (E(I-286)), determined from the pH dependence of the F-formation kinetics, was found to be 9.4, while the pK(a) of the proton acceptor at the catalytic site is likely to be > or = 2.5 pH units higher. In the pH range up to pH 10 the proton equilibrium between the bulk solution and E(I-286) was much faster than 10(4) s(-1), while in the pH range above pH 10 the proton uptake from solution is rate limiting for the overall reaction. The apparent second-order rate constant for proton transfer from the bulk solution to E(I-286) is >10(13) M(-1) s(-1), which indicates that the proton uptake is assisted by a local buffer consisting of protonatable residues at the protein surface.