シトクロムcオキシダーゼ (CCO) または複合体IVは、バクテリアおよびミトコンドリアで見られる膜貫通タンパク質複合体の一つで、膜における電子伝達系の最後の酵素である。4分子のシトクロムcから電子を受け取り、酸素1分子に転移させ、2分子の水に変換する機能を持つ。この過程では、マトリックス由来の4個のプロトンから水が生成されると同時に、4個のプロトンがマトリックスから膜間スペースに透過する。これにより発生した膜間の電気化学ポテンシャルの差がATP合成酵素によるATP合成に用いられる。
（クリックで拡大）
図1 ウシシトクロムcオキシダーゼ の結晶構造（PDBID:１OCC）
複合体IVはいくつかの金属補欠分子族部位と13のタンパク質サブユニットから構成される巨大な内在性膜タンパク質である。これには2種のヘム（ヘムa 、ヘムa 3）、2種の銅中心（CuAとCuB）が含まれている。2種類のヘムとCuBはサブユニットIに位置し、2個のCuAはサブユニットIIに配位している。サブユニットIのヘムa 3とCuBはそれぞれで二核中心を形成し、酸素の還元部位となっている。シトクロムc は複合体IIIのシトクロムc 1によって還元された後、複合体IVのCuA二核中心と結合し、シトクロムc の鉄中心はFe2+からFe3+に酸化される。還元されたCuA二核中心はその電子をヘムa に送り、さらにそこからヘムa 3-CuB二核中心に送られる。この二核中心の2個の金属イオンは4.5 Å離れており、十分な酸化状態の水酸化物イオンに配位している。シトクロムc はTyr(244)のC6とHis(240)のε-Nが結合するという独特な翻訳後修飾が見られ、、ヘムa 3-CuB二核中心が4電子を受け取って酸素分子を水に還元するという極めて重要な役割が可能になっている。以前は、還元機構は過酸化物中間体が関与していると考えられ、それが超酸化物の生成に繋がっていると考えられていた。しかし、現代では、4電子還元によって酸素-酸素結合が開裂する反応機構が支持されており、超酸化物が形成しそうな中間体は避けられている。
反応の概要：
4 Fe2+-シトクロム c + 8 H+in + O2 → 4 Fe3+-シトクロム c + 2 H2O + 4 H+out
2個の電子がシトクロムcからCuA二核中心とヘムa を通過して、ヘムa 3-CuB二核中心に至り、このFe3+はFe2+に、Cu2+はCu+に還元される。このときそれぞれの金属イオンに配位していたヒドロキシル配位子はプロトン化されて水として失われ、金属間に酸素分子が入る空間が作られる。酸素はFe2+-シトクロム c由来の2電子により迅速に還元され、フェリオキソ型(Fe+4=O)に変換される。CuB側の酸素原子はCu+からの1電子と、Tyr(244)の由来の1電子と1プロトンを受け取りヒドロキシ配位子に変換される。このときTyr(244)はチロシルラジカルとなる。別のシトクロムc から発生する3番目の電子は始めの2種の電子キャリアーからヘムa 3-CuBに至り、この電子と2プロトンによりチロシルラジカルがチロシンに戻り、そしてヒドロキシドはCuB2+に結合し後に水分子となる。同様に4番目の電子も始めの2種の電子キャリアーからヘムa 3-CuBに至ることによりFe+4=OがFe+3に還元され、同時に酸素原子がプロトンを受け取り、ヘムa 3-CuBがこのサイクルの始めの状態に戻る。
図2 CCOの活性中心におけるプロトンと電子の流れ。
”Two tyrosyl radicals stabilize high oxidation states in cytochrome C oxidase for efficient energy conservation and proton translocation”.J Am Chem Soc.2012 Mar 14;134(10):4753-61.Yu MA, Egawa T, Shinzawa-Itoh K, Yoshikawa S, Guallar V, Yeh SR, Rousseau DL, Gerfen GJ.
The reaction of oxidized bovine cytochrome c oxidase (bCcO) with hydrogen peroxide (H(2)O(2)) was studied by electron paramagnetic resonance (EPR) to determine the properties of radical intermediates. Two distinct radicals with widths of 12 and 46 G are directly observed by X-band EPR in the reaction of bCcO with H(2)O(2) at pH 6 and pH 8. High-frequency EPR (D-band) provides assignments to tyrosine for both radicals based on well-resolved g-tensors. The wide radical (46 G) exhibits g-values similar to a radical generated on L-Tyr by UV-irradiation and to tyrosyl radicals identified in many other enzyme systems. In contrast, the g-values of the narrow radical (12 G) deviate from L-Tyr in a trend akin to the radicals on tyrosines with substitutions at the ortho position. X-band EPR demonstrates that the two tyrosyl radicals differ in the orientation of their β-methylene protons. The 12 G wide radical has minimal hyperfine structure and can be fit using parameters unique to the post-translationally modified Y244 in bCcO. The 46 G wide radical has extensive hyperfine structure and can be fit with parameters consistent with Y129. The results are supported by mixed quantum mechanics and molecular mechanics calculations. In addition to providing spectroscopic evidence of a radical formed on the post-translationally modified tyrosine in CcO, this study resolves the much debated controversy of whether the wide radical seen at low pH in the bovine enzyme is a tyrosine or tryptophan. The possible role of radical formation and migration in proton translocation is discussed.
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