Conformational coupling between the active site and residues within the KC-channel of the Vibrio cholerae cbb3-type (C-family) oxygen reductase

Abstract

The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the KC-channel is a conserved glutamate in subunit III. However, the majority of the KC-channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the KC-channel, was found to depend on the conformation of Y241Vc, located in subunit I at the interface with subunit III. Mutations of Y241Vc (to A/F/H/S) in the Vibrio cholerae cbb3 eliminate catalytic activity, but also cause perturbations that propagate over a 28-A˚ distance to the active site heme b3. The data suggest a linkage between residues lining the KC-channel and the active site of the enzyme, possibly mediated by transmembrane helix α7, which contains both Y241Vc and the active site cross-linked Y255Vc, as well as two CuB histidine ligands. Other mutations of residues within or near helix α7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme.