Alternative initial proton acceptors for the D pathway of Rhodobacter sphaeroides cytochrome c oxidase.

To characterize protein structures that control proton uptake, we assayed forms of cytochrome c oxidase (CcO) containing a carboxyl or a thiol group in line with the initial, internal waters of the D pathway for proton transfer in the presence and absence of subunit III. Subunit III provides approximately half of the protein surrounding the entry region of the D pathway. The N139D/D132N mutant contains a carboxyl group 6 Å within the D pathway and lacks the normal, surface-exposed proton acceptor, Asp-132. With subunit III, the steady-state activity of this mutant is slow, but once subunit III is removed, its activity is the same as that of wild-type CcO lacking subunit III (∼1800 H+/s). Thus, a carboxyl group∼25% within the pathway enhances proton uptake even though the carboxyl has no direct contact with bulk solvent. Protons from solvent apparently move to internal Asp-139 through a short file of waters, normally blocked by subunit III. Cys-139 also supports rapid steady-state proton uptake, demonstrating that an anion other than a carboxyl can attract and transfer protons into the D pathway. When both Asp-132 and Asp/Cys-139 are present, the removal of subunit III increases CcO activity to rates greater than that of normal CcO because of simultaneous proton uptake by two initial acceptors. The results show how the environment of the initial proton acceptor for the D pathway in these CcO forms dictates the pH range of CcO activity, with implications for the function of Asp-132, the normal proton acceptor.