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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 106; no. 7; pp. 2165 - 2169
• Main Authors: Aoyama, Hiroshi, Muramoto, Kazumasa, Shinzawa-Itoh, Kyoko, Hirata, Kunio, Yamashita, Eiki, Tsukihara, Tomitake, Ogura, Takashi, Yoshikawa, Shinya
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 17.02.2009; National Acad Sciences
• Subjects: Animals; Binding Sites; Biological Sciences; Cattle; Copper - chemistry; Crystallography, X-Ray - methods; Crystals; Cytochromes; Datasets; Electron density; Electron Transport Complex IV - chemistry; Electrons; Ions; Iron - chemistry; Irradiation; Metals - chemistry; Molecules; Oxidases; Oxygen - chemistry; Peroxides; Peroxides - chemistry; Physical Sciences; Proton Pumps - chemistry; Protons; Spectrum Analysis, Raman; Ungulates; X-Rays
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0806391106

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-Å resolution by limiting the X-ray dose for each shot and by using many ([almost equal to]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 O₂ reduction site (Fe³⁺-O⁻-O⁻-Cu²⁺), in contrast to a ferric hydroxide (Fe³⁺-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 O₂ reduction site suppresses the proton pumping function.