Kinetic gating of the proton pump in cytochrome c oxidase

Abstract

Cytochrome c oxidase (CcO), the terminal enzyme of the respiratory chain, reduces oxygen to water and uses the released energy to pump protons across a membrane. Here, we use kinetic master equations to explore the energetic and kinetic control of proton pumping in CcO. We construct models consistent with thermodynamic principles, the structure of CcO, experimentally known proton affinities, and equilibrium constants of intermediate reactions. The resulting models are found to capture key properties of CcO, including the midpoint redox potentials of the metal centers and the electron transfer rates. We find that coarse-grained models with two proton sites and one electron site can pump one proton per electron against membrane potentials exceeding 100 mV. The high pumping efficiency of these models requires strong electrostatic couplings between the proton loading (pump) site and the electron site (heme a), and kinetic gating of the internal proton transfer. Gating is achieved by enhancing the rate of proton transfer from the conserved Glu-242 to the pump site on reduction of heme a, consistent with the predictions of the water-gated model of proton pumping. The model also accounts for the phenotype of D-channel mutations associated with loss of pumping but retained turnover. The fundamental mechanism identified here for the efficient conversion of chemical energy into an electrochemical potential should prove relevant also for other molecular machines and novel fuel-cell designs.bioenergetics ͉ biological machines ͉ kinetic master equation ͉ respiration C ytochrome c oxidase (CcO) captures the energy from the reduction of oxygen to drive the translocation of protons across the inner mitochondrial (or bacterial) membrane (1-8). The resulting electrochemical gradient powers the production of ATP, the energy source of the cell. CcO takes up four electrons from the outside (P side) of the membrane and four protons from the inside (N side) for the reduction of one dioxygen molecule. Four additional protons are translocated across the membrane from the N side to the P side. Crystal structures of the enzyme from various organisms (3, 4, 9, 10) indicate the electron and proton pathways, formed by conserved residues and cofactors. Extensive experimental and theoretical studies have provided valuable information about these pathways, the reaction sequence, and energetic aspects of catalysis (5,7,9,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). However, some key aspects of the fundamental mechanisms governing the coupling between the chemical reaction and vectorial proton translocation are still unclear.Here, the objective is to explain how vectorial proton translocation is accomplished in CcO, with respect to both the driving forces (i.e., proton and electron affinities expressed as pK a values and midpoint redox potentials, respectively, and Coulombic couplings) and the kinetic control (i.e., the ''gating'' effects that result from a dependence of kinetic barriers on the microscopic states). Specifically, we aim to construct ...

Reversibility and efficiency in electrocatalytic energy conversion and lessons from enzymes

“…Conversely, some enzymes have evolved to act irreversibly, to prevent back-reactions and maintain fluxes through pathways. Cytochrome c oxidase is the last enzyme in the mitochondrial respiratory chain; it reduces O 2 to water in a series of stepwise electron-proton transfers that are tightly coupled to proton translocation (40). Only part of the energy from this reaction is conserved in proton pumping, and even the largest proton-motive forces available to biology are unable to drive the reverse reaction.…”

Energy transducing redox steps of the Na + -pumping NADH:quinone oxidoreductase from Vibrio cholerae

“…Although the coupling mechanism of most of the respiratory chain complexes and other redox pumps have been studied in detail (15)(16)(17), the fundamental mechanism of cation pumping in Na þ -NQR might follow different rules because this is the only known example of a redox pump that translocates sodium instead of protons. The elucidation of this mechanism may open new windows for the understanding of cation pumping in other enzymes and could help to define general principles of biological transport as a whole.…”

Toward molecular models of proton pumping: Challenges, methods and relevant applications

“…Thus understanding factors that dictate the rate of proton transfers in complex systems is crucial [12,15,83]. The fact that proton transfer inherently involves bondbreaking and bond-making calls for the use of a quantum mechanics (QM) based method for modeling the process at the microscopic level.…”