Electrostatic control of proton pumping in cytochrome c oxidase

Abstract

As part of the mitochondrial respiratory chain, cytochrome c oxidase utilizes the energy produced by the reduction of O2 to water to fuel vectorial proton transport. The mechanism coupling proton pumping to redox chemistry is unknown. Recent advances have provided evidence that each of the four observable transitions in the complex catalytic cycle consists of a similar sequence of events. However, the physico-chemical basis underlying this recurring sequence has not been identified. We identify this recurring pattern based on a comprehensive model of the catalytic cycle derived from the analysis of oxygen chemistry and available experimental evidence. The catalytic cycle involves the periodic repetition of a sequence of three states differing in the spatial distribution of charge in the active site: [0|1], [1|0], and [1|1], where the total charge of heme a and the binuclear center appears on the left and on the right, respectively. This sequence recurs four times per turnover despite differences in the redox chemistry. This model leads to a simple, robust, and reproducible sequence of electron and proton transfer steps and rationalizes the pumping mechanism in terms of electrostatic coupling of proton translocation to redox chemistry. Continuum electrostatic calculations support the proposed mechanism and suggest an electrostatic origin for the decoupled and inactive phenotypes of ionic mutants in the principal proton-uptake pathway.

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

“…The above results shed light onto the role of residues lining the D channel in the uptake and relay of protons to the active site of the enzyme. These findings are consistent with our previous analysis of structural fluctuations in the D channel [11] and support our previously proposed electrostatic model of proton pumping [32]. Detailed free energy simulations of the conformational equilibrium of residue 139 confirm that the closed conformer found in crystal structures of the enzyme is the lowest energy state in both the wild-type and the N139D mutant.…”

“…As previously discussed [32], the introduction of a negatively charged residue at position 139 destabilizes the ionic state of residue E286 relative to its neutral, protonated state, resulting in a further increase in its already elevated proton affinity. This perturbation is significant in the closed conformer of N139D, which is the most populated state of the single mutant.…”

“…The charge distribution of the binuclear centre was calculated as described in Fadda et al [32]. The enzyme was simulated in the fully reduced R state.…”

“…Ten catalytic states denoted F through MV are shown, which are thought to involve the uptake of protons through the D channel, as described in detail in Fadda et al [32]. Together, these ten distinct catalytic states make up three out of four recurring subcycles in which electron and proton transfer are coupled electrostatically: the uptake of a proton by the PLS following reduction of heme a in state [0|1], the uptake of a chemical proton by the BNC following electron transfer from heme a to heme a3 in state [1|0], and the pumping of a proton upon the ensuing maximization of the positive charge in the active site of the enzyme (state [1|1]) [32]. As seen in figure 4, the periodic recurrence of the three charge states induces a pseudo-periodic cycle in the pK a of E286, the residue at the top of the D channel which shuttles protons from the D channel alternatively to the PLS and to the BNC in states [0|1] and [1|0], respectively.…”

“…However, the mechanism which couples redox chemistry and proton pumping remains unclear. Recently, we proposed a model for the catalytic cycle of CcO which rests on the control of vectorial proton transfer by long-range electrostatic interactions in a recurring sequence of electron and proton transfer steps occurring in the active site [32]. In this model, E286 plays a vital role by relaying most of the chemical and pumped protons to the BNC and the PLS, respectively.…”