Structural Elements Involved in Proton Translocation by CytochromecOxidase as Revealed by Backbone Amide Hydrogen−Deuterium Exchange of the E286H Mutant

Abstract

Cytochrome c oxidase is the terminal electron acceptor in the respiratory chains of aerobic organisms and energetically couples the reduction of oxygen to water to proton pumping across the membrane. The mechanisms of proton uptake, gating, and pumping have yet to be completely elucidated at the molecular level for these enzymes. For Rhodobacter sphaeroides CytcO (cytochrome aa3), it appears as though the E286 side chain of subunit I is a branching point from which protons are shuttled either to the catalytic site for O2 reduction or to the acceptor site for pumped protons. Amide hydrogen-deuterium exchange mass spectrometry was used to investigate how mutation of this key branching residue to histidine (E286H) affects the structures and dynamics of four redox intermediate states. A functional characterization of this mutant reveals that E286H CytcO retains approximately 1% steady-state activity that is uncoupled from proton pumping and that proton transfer from H286 is significantly slowed. Backbone amide H-D exchange kinetics indicates that specific regions of CytcO, perturbed by the E286H mutation, are likely to be involved in proton gating and in the exit pathway for pumped protons. The results indicate that redox-dependent conformational changes around E286 are essential for internal proton transfer. E286H CytcO, however, is incapable of these specific conformational changes and therefore is insensitive to the redox state of the enzyme. These data support a model where the side chain conformation of E286 controls proton translocation in CytcO through its interactions with the proton gate, which directs the flow of protons either to the active site or to the exit pathway. In the E286H mutant, the proton gate does not function properly and the exit channel is unresponsive. These results provide new insight into the structure and mechanism of proton translocation by CytcO.

A D-Pathway Mutation Decouples the Paracoccus denitrificans Cytochrome c Oxidase by Altering the Side-Chain Orientation of a Distant Conserved Glutamate

“…Results from several studies 3,[32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] indicate that the side chain of Glu278, located at the upper end of the D-pathway, may be the key element for gating. From Glu278, protons are funneled into two sites (see Fig.…”

Location of Inhibitor Binding Sites in the Human Inducible Prostaglandin E Synthase, MPGES1

“…11-17 In the last decade the technique has been successfully applied to the analysis of ligand binding to, and the conformational dynamics of integral membrane proteins including members of the MAPEG superfamily. 18-21 In this report we demonstrate the utility of H/D exchange mass spectrometry (MS) to locate specific inhibitor binding sites on the integral membrane protein, human MPGES1. The results indicate that there are unique features that distinguish different types of inhibitors and common attributes among some inhibitors discovered by medicinal chemistry efforts.…”

Functional interactions between membrane-bound transporters and membranes

“…It has also been suggested that Glu II 101 may act as a gate controlling proton delivery into the K pathway (29). Furthermore, it has been shown that structural changes in the K pathway are orchestrated with transitions between catalytic intermediates in CytcO (30)(31)(32), which suggests that functional interactions between this pathway and the membrane are important for understanding function.…”