The K Proton-Channel of C-Family O2 Reductase from Vibrio Cholerae

Abstract

The heme-copper oxygen reductases are integral membrane proteins that reduce O2 to water, producing an electrochemical proton gradient across the membrane. Nearly all organisms that depend on aerobic respiration have a proper functioning heme-copper oxygen reductase for their survival. There are 3 major families of these enzymes, called the A, B and C-families. Several human pathogens contain a C-family O2 reductase, also known as a cbb3 oxidase, as the lone oxygen reductase in their respiratory chains. This enzyme is, therefore, an attractive drug target. X-ray crystallography and site-directed mutagenesis studies have shown that C-family O2 reductase contains only one proton input channel (K-channel analogue) to transfer protons for O2 reduction chemistry as well as proton pumping. In this work we examine mutations in the K-channel analogue of the cbb3 oxygen reductase from Vibrio cholerae with particular focus on identification of the entrance of the channel. The X-ray structure of the cbb3 oxidase from Pseudomonas stutzeri indicates that the highly conserved glutamic acid E49III (V. cholerae numbering) within CcoP (subunit III) is a plausible candidate for the entrance of the K-channel analogue. It is shown in the current work that the replacement of this glutamic acid by alanine (E49IIIA) strongly inhibits oxidase activity. This indicates that the entrance of the K-channel analogue of the cbb3 oxygen reductase from V. cholerae is the surface-exposed glutamic acid E49 in CcoP. Following this entrance residue, the majority of residues lining the K-channel analogue are located in subunit I. Mutations in many of these residues also inhibit oxygen reductase activity.