Molecular basis of proton uptake in single and double mutants of cytochromecoxidase

Abstract

Cytochrome c oxidase, the terminal enzyme of the respiratory chain, utilizes the reduction of dioxygen into water to pump protons across the mitochondrial inner membrane. The principal pathway of proton uptake into the enzyme, the D channel, is a 2.5 nm long channel-like cavity named after a conserved, negatively charged aspartic acid (D) residue thought to help recruiting protons to its entrance (D132 in the first subunit of the S. sphaeroides enzyme). The single-point mutation of D132 to asparagine (N), a neutral residue, abolishes enzyme activity. Conversely, replacing conserved N139, one-third into the D channel, by D, induces a decoupled phenotype, whereby oxygen reduction proceeds but not proton pumping. Intriguingly, the double mutant D132N/N139D, which conserves the charge of the D channel, restores the wild-type phenotype. We use molecular dynamics simulations and electrostatic calculations to examine the structural and physical basis for the coupling of proton pumping and oxygen chemistry in single and double N139D mutants. The potential of mean force for the conformational isomerization of N139 and N139D side chains reveals the presence of three rotamers, one of which faces the channel entrance. This out-facing conformer is metastable in the wild-type and in the N139D single mutant, but predominant in the double mutant thanks to the loss of electrostatic repulsion with the carboxylate group of D132. The effects of mutations and conformational isomerization on the pKa of E286, an essential proton-shuttling residue located at the top of the D channel, are shown to be consistent with the electrostatic control of proton pumping proposed recently (Fadda et al 2008 Biochim. Biophys. Acta 1777. Taken together, these results suggest that preserving the spatial distribution of charges at the entrance of the D channel is necessary to guarantee both the uptake and the relay of protons to the active site of the enzyme. These findings highlight the interplay of long-range electrostatic forces and local structural fluctuations in the control of proton movement and provide a physical explanation for the restoration of proton pumping activity in the double mutant.

Exploring the Possible Role of Glu286 in CcO by Electrostatic Energy Computations Combined with Molecular Dynamics

“…Fine tuning of the pK A of Glu286 seems to be crucial for the proton pump mechanism of CcO. Mutants abolishing CcO function have been suggested to downshift the pK A of Glu286 49,50 . It is reasonable to assume that the pK A of Glu286 needs to be above the pK A of the resting PLS to store a proton, but below the pK A value of the activated PLS to donate a proton within the pump cycle.…”

Role of aspartate 132 at the orifice of a proton pathway in cytochromecoxidase

“…These data were interpreted to suggest that slowed proton uptake in the D132N single mutant is caused by the removal of negative charge (40). Proton uptake in the D132A CytcO also could be partly restored upon removal of SUIII, which eliminates a significant part of the residues surrounding the D-pathway orifice and presumably allows water molecules to access the proton pathway (38).…”

Multiscale simulations reveal key features of the proton pumping mechanism in cytochromecoxidase

“…Three highly conserved asparagine residues, N139, N121, and N207, reside roughly one third of the way into the D-channel and form a constricted region (called the asparagine gate) (36). Previous nonreactive classical MD simulations have revealed a gating motion of N139 that controls the hydration state of D-channel (37,38).…”