Proton input channel of cytochrome cbb(3) type oxidase in Rhodobacter capsulatus

Cytochrome cbb3 is a distinct member of the superfamily of respiratory heme-copper oxidases responsible for catalyzing the reduction of oxygen to water. As in all A-family, B-family, and C-family this reaction drives proton pumping across the membrane. Mutagenesis studies on family A have identified two conserved proton input channels: D and K. The residues in the A-family that are located in the proton channels are not conserved in the C-family. Since the C-type oxygen reductases have been shown to pump protons, presence of proton-conducting channels is obvious in this family that play analogous roles to the D-channel and/or K-channel. In this work, sequences were aligned to identify functionally important residues which may form the proton channels in the C-family. The functional importance of the residues was tested by site-directed mutagenesis using the cbb3 oxidase from Rhodobacter capsulatus. Several residues proposed to be part of the putative K-channel had significantly reduced catalytic activity upon mutations: T272A, Y280F/Y281F, T319M, N346V, and Y374F. The results support the proposal that in the C-family only one channel, analogous to the K-channel in the A-type oxygen reductases, functions for the delivery of both catalytic and pumped protons.