Ribacka Camilla, Verkhovsky Michael I, Belevich Ilya, Bloch Dmitry A, Puustinen Anne, Wikström Mårten
Helsinki Bioenergetics Group, Program for Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, PB 65 (Viikinkaari 1), FIN-00014, Helsinki, Finland.
Biochemistry. 2005 Dec 20;44(50):16502-12. doi: 10.1021/bi0511336.
Cytochrome c oxidase couples reduction of dioxygen to water to translocation of protons over the inner mitochondrial or bacterial membrane. A likely proton acceptor for pumped protons is the Delta-propionate of heme a(3), which may receive the proton via water molecules from a conserved glutamic acid (E278 in subunit I of the Paracoccus denitrificans enzyme) and which receives a hydrogen bond from a conserved tryptophan, W164. Here, W164 was mutated to phenylalanine (W164F) to further explore the role of the heme a(3) Delta-propionate in proton translocation. FTIR spectroscopy showed changes in vibrations possibly attributable to heme propionates, and the midpoint redox potential of heme a(3) decreased by approximately 50 mV. The reaction of the oxidized W164F enzyme with hydrogen peroxide yielded substantial amounts of the intermediate F' even at high pH, which suggests that the mutation rearranges the local electric field in the binuclear center that controls the peroxide reaction. The steady-state proton translocation stoichiometry of the W164F enzyme dropped to approximately 0.5 H(+)/e(-) in cells and reconstituted proteoliposomes. Time-resolved electrometric measurements showed that when the fully reduced W164F enzyme reacted with O(2), the membrane potential generated in the fast phase of this reaction was far too small to account either for full proton pumping or uptake of a substrate proton from the inside of the proteoliposomes. Time-resolved optical spectroscopy showed that this fast electrometric phase occurred with kinetics corresponding to the transition from state A to P(R), whereas the subsequent transition to the F state was strongly delayed. This is due to a delay of reprotonation of E278 via the D-pathway, which was confirmed by observation of a slowed rate of Cu(A) oxidation and which explains the small amplitude of the fast charge transfer phase. Surprisingly, the W164F mutation thus mimics a weak block of the D-pathway, which is interpreted as an effect on the side chain isomerization of E278. The fast charge translocation may be due to transfer of a proton from E278 to a "pump site" above the heme groups and is likely to occur also in wild-type enzyme, though not distinguished earlier due to the high-amplitude membrane potential formation during the P(R)--> F transition.
细胞色素c氧化酶将氧气还原为水的过程与质子在线粒体内膜或细菌膜上的跨膜转运相偶联。被泵出的质子的一个可能的受体是血红素a(3)的δ-丙酸酯，它可能通过水分子从一个保守的谷氨酸（反硝化副球菌酶亚基I中的E278）接收质子，并且它从一个保守的色氨酸W164接受氢键。在此，将W164突变为苯丙氨酸（W164F）以进一步探索血红素a(3)δ-丙酸酯在质子转运中的作用。傅里叶变换红外光谱显示振动变化可能归因于血红素丙酸酯，并且血红素a(3)的中点氧化还原电位降低了约50 mV。即使在高pH值下，氧化型W164F酶与过氧化氢的反应也产生大量的中间体F'，这表明该突变重新排列了控制过氧化物反应的双核中心的局部电场。在细胞和重组蛋白脂质体中，W164F酶的稳态质子转运化学计量比降至约0.5 H(+)/e(-)。时间分辨的电测量表明，当完全还原的W164F酶与O(2)反应时，该反应快速阶段产生的膜电位太小，无法解释完全的质子泵浦或从蛋白脂质体内部摄取底物质子。时间分辨光谱表明，这个快速电测量阶段的动力学与从状态A到P(R)的转变相对应，而随后向F状态的转变则被强烈延迟。这是由于通过D途径对E278的再质子化延迟，这通过观察到Cu(A)氧化速率减慢得到证实，并且这解释了快速电荷转移阶段的小幅度。令人惊讶的是，W164F突变因此模拟了D途径的弱阻断，这被解释为对E278侧链异构化的影响。快速电荷转运可能是由于质子从E278转移到血红素基团上方的“泵位点”，并且可能也发生在野生型酶中，尽管由于在P(R)--> F转变期间形成的高幅度膜电位而未被早期区分。
