Molecular basis of proton uptake in single and double mutants of cytochromec oxidase

Abstract

Cytochrome c oxidase, the terminal enzyme of the respiratory chain, utilizes the reduction of dioxygeninto water to pump protons across the mitochondrial inner membrane. The principalpathway of proton uptake into the enzyme, the D channel, is a 2.5 nm long channel-likecavity named after a conserved, negatively charged aspartic acid (D) residue thought tohelp recruiting protons to its entrance (D132 in the first subunit of the S. sphaeroidesenzyme). The single-point mutation of D132 to asparagine (N), a neutral residue,abolishes enzyme activity. Conversely, replacing conserved N139, one-third into the Dchannel, by D, induces a decoupled phenotype, whereby oxygen reduction proceedsbut not proton pumping. Intriguingly, the double mutant D132N/N139D, whichconserves the charge of the D channel, restores the wild-type phenotype. We usemolecular dynamics simulations and electrostatic calculations to examine thestructural and physical basis for the coupling of proton pumping and oxygenchemistry in single and double N139D mutants. The potential of mean force for theconformational isomerization of N139 and N139D side chains reveals the presence of threerotamers, one of which faces the channel entrance. This out-facing conformer ismetastable in the wild-type and in the N139D single mutant, but predominant in thedouble mutant thanks to the loss of electrostatic repulsion with the carboxylategroup of D132. The effects of mutations and conformational isomerization on thepKa of E286, an essential proton-shuttling residue located at the top of the D channel, areshown to be consistent with the electrostatic control of proton pumping proposed recently(Fadda et al 2008 Biochim. Biophys. Acta 1777 277–84). Taken together, these resultssuggest that preserving the spatial distribution of charges at the entrance of the D channelis necessary to guarantee both the uptake and the relay of protons to the active site of theenzyme. These findings highlight the interplay of long-range electrostatic forces and localstructural fluctuations in the control of proton movement and provide a physicalexplanation for the restoration of proton pumping activity in the double mutant.