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Cytochrome c oxidase

Proton pumping steps during catalytic cycle
From the discovery of cytochromes by David Keilin in the 1920s1 it took more than 40 years of investigation2 to find that CcO conserves energy by maintaining a ΔμH on a membrane, by virtue of the vectorial organization of its chemistry, and 10 more years to discover that for maximal efficiency the enzyme can perform active transport of protons from one side of the membrane dielectric to the other (pumping)3.

Results of numerous studies have been developed into a symmetrical scheme of the pumping events during the catalytic cycle of CcO. Under continuous turnover conditions CcO proceeds via four relatively stable intermediates: PM, F, OH, and EH. Single electron reduction of each of these four intermediates from cytochrome c leads to uptake of a "chemical" proton from the N-side with simultaneous translocation of another proton across the membrane.

The proton translocation during the catalytic cycle of cytochrome c oxidase

The proton translocation during the catalytic cycle of cytochrome c oxidase. The active states are shown in squares. The transitions between each of them are coupled to proton pumping. The relaxed states are shown in circles; their reduction is not coupled to proton pumping.

Model of Single Proton Translocation Cycle4
During single-electron reduction of the oxidase, the electron from cytochrome c first enters the CuA center.

In the next phase the electron equilibrates between CuA and heme a, with time constant ~10 μs (I). The arrival of electron on heme a raises the pKa of a yet unidentified “pump site” above the heme groups, which takes up a proton with τ ~ 150 μs. The protonation of the "pump site" occurs from the conserved GluI278 via a chain of water molecules by the Grotthuss mechanism. Three or four water molecules are predicted to reside inside the hydrophobic cavity between the glutamate, the Δ-propionate of heme a3, and the binuclear center. These water molecules are sensitive to the redox-state-dependent electric field between heme a and the binuclear center, and arrange themselves into two different configurations for proton transfer and chemistry. When the binuclear center is oxidized and the electron is on heme a, the array of water molecules is oriented towards the Δ-propionate of heme a3 ("pump site" direction); however this array switches towards the binuclear center (the direction for chemical reaction) after reduction of the binuclear center from heme a.

The rate-limiting protonation of the "pump site" raises the Em of both hemes, and leads to further electron equilibration between CuA and the two hemes in the same time window (II). At the end of the 150 μs phase CuA is fully oxidized, while heme a and heme a3 have 40% and 60% of the injected electron population respectively. In addition, the 150 μs phase includes reprotonation of GluI278 from the N-side of the membrane via the proton conducting D-pathway.

Single proton translocation cycle, part I. Electron transfer shown with blue, and proton transfer with red arrows.

Single proton translocation cycle, part I. Electron transfer shown with blue, and proton transfer with red arrows.

In the next phase (τ ~ 800 μs), transfer of a substrate proton to the OH- ligand of CuB raises the Em of CuB to a value much higher than those of all other redox centers, which induces ultimate movement of the electron to the CuB center (III).

During the last step of the single proton pump cycle, the proton, which has been "preloaded" into the "pump site", is expelled towards the P-side of the membrane due to electrostatic repulsion from the substrate proton (IV).

Single proton translocation cycle, part II

Single proton translocation cycle, part II.

It seems feasible that the mechanism of proton translocation during each of the PM->F, F->OH, and EH->R transitions is essentially the same. There are though some differences such as the final destination of the electron (CuB, or heme a3), or the exact channel (D, or K) used by chemical proton.

Thus, the sequence of events presented in the model would be repeated every time an electron enters the CuA site. In each case the electron travels from cytochrome c to CuA, and subsequently through heme a to the binuclear center, driving proton pumping across the membrane.


References
1. Keilin D. On cytochrome, a respiratory pigment common to animals, yeast, and higher plants. // Proc. R. Soc. Lond. B Biol. Sci., 1925, 98, 312–339.
2. Slater EC. Keilin, cytochrome, and the respiratory chain. // J. Biol. Chem., 2003, 278, 16455–16461 [link].
3. Wikström MK. Proton pump coupled to cytochrome c oxidase in mitochondria. // Nature, 1977, 266, 271–273 [link].
4. Belevich I, Bloch DA, Belevich N, Wikström M, Verkhovsky MI. Exploring the proton pump mechanism of cytochrome c oxidase in real time. // Proc. Nat. Acad. Sci. USA, 2007, 104, 2685-2690 [link].


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