A peroxide bridge between Fe and Cu ions in the O2 reduction site of fully oxidized cytochrome c oxidase could suppress the proton pump.

The fully oxidized form of cytochrome c oxidase, immediately after complete oxidation of the fully reduced form, pumps protons upon each of the initial 2 single-electron reduction steps, whereas protons are not pumped during single-electron reduction of the fully oxidized "as-isolated" form (the fully oxidized form without any reduction/oxidation treatment) [Bloch D, et al. (2004) The catalytic cycle of cytochrome c oxidase is not the sum of its two halves. Proc Natl Acad Sci USA 101:529-533]. For identification of structural differences causing the remarkable functional difference between these 2 distinct fully oxidized forms, the X-ray structure of the fully oxidized as-isolated bovine heart cytochrome c oxidase was determined at 1.95-A resolution by limiting the X-ray dose for each shot and by using many (approximately 400) single crystals. This minimizes the effects of hydrated electrons induced by the X-ray irradiation. The X-ray structure showed a peroxide group bridging the 2 metal sites in the O(2) reduction site (Fe(3+)-O(-)-O(-)-Cu(2+)), in contrast to a ferric hydroxide (Fe(3+)-OH(-)) in the fully oxidized form immediately after complete oxidation from the fully reduced form, as has been revealed by resonance Raman analyses. The peroxide-bridged structure is consistent with the reductive titration results showing that 6 electron equivalents are required for complete reduction of the fully oxidized as-isolated form. The structural difference between the 2 fully oxidized forms suggests that the bound peroxide in the O(2) reduction site suppresses the proton pumping function.