Verkhovsky M I, Jasaitis A, Verkhovskaya M L, Morgan J E, Wikström M
Helsinki Bioenergetics Group, Department of Medical Chemistry, Institute of Biomedical Sciences and Biocentrum Helsinki, University of Helsinki, Finland.
Nature. 1999 Jul 29;400(6743):480-3. doi: 10.1038/22813.
Cell respiration in mitochondria and some bacteria is catalysed by cytochrome c oxidase, which reduces O2 to water, coupled with translocation of four protons across the mitochondrial or bacterial membrane. The enzyme's catalytic cycle consists of a reductive phase, in which the oxidized enzyme receives electrons from cytochrome c, and an oxidative phase, in which the reduced enzyme is oxidized by O2. Previous studies indicated that proton translocation is coupled energetically only to the oxidative phase, but this has been challenged. Here, with the purified enzyme inlaid in liposomes, we report time-resolved measurements of membrane potential, which show that half of the electrical charges due to proton-pumping actually cross the membrane during reduction after a preceding oxidative phase. pH measurements confirm that proton translocation also occurs during reduction, but only when immediately preceded by an oxidative phase. We conclude that all the energy for proton translocation is conserved in the enzyme during its oxidation by O2. One half of it is utilized for proton-pumping during oxidation, but the other half is unlatched for this purpose only during re-reduction of the enzyme.
线粒体和某些细菌中的细胞呼吸由细胞色素c氧化酶催化，该酶将O2还原为水，并伴随着四个质子跨线粒体或细菌膜的转运。该酶的催化循环包括一个还原阶段，在此阶段氧化态的酶从细胞色素c接收电子；以及一个氧化阶段，在此阶段还原态的酶被O2氧化。先前的研究表明质子转运仅在能量上与氧化阶段偶联，但这一观点受到了挑战。在这里，我们将纯化的酶嵌入脂质体中，报告了膜电位的时间分辨测量结果，结果表明在前一个氧化阶段之后的还原过程中，由于质子泵作用产生的一半电荷实际上穿过了膜。pH测量证实质子转运在还原过程中也会发生，但只有在紧接氧化阶段之后才会发生。我们得出结论，质子转运的所有能量在酶被O2氧化的过程中都保存在酶中。其中一半能量在氧化过程中用于质子泵作用，但另一半能量只有在酶再次还原时才会被释放用于此目的。
