Verkhovsky M I, Tuukkanen A, Backgren C, Puustinen A, Wikström M
Department of Medical Chemistry, Institute of Biomedical Sciences and Biocentrum Helsinki, P.O. Box 8, 00014 University of Helsinki, Helsinki, Finland.
Biochemistry. 2001 Jun 19;40(24):7077-83. doi: 10.1021/bi010030u.
Electrons were discretely injected into oxidized cytochrome c oxidase in liposomes by laser flash excitation of bound ruthenium [II] bispyridyl, and the membrane potential was recorded by time-resolved electrometry. Membrane potential is generated in a fast phase when an electron is transferred from the excited dye, via the CuA center, to heme a at a relative dielectric depth d inside the membrane [Zaslavsky, D., Kaulen, A. D., Smirnova, I. A., Vygodina, T., and Konstantinov, A. A. (1993) FEBS Lett. 336, 389-393]. Subsequently, membrane potential may develop further in a slower event, which is due to proton transfer into the enzyme from the opposite side of the membrane [Ruitenberg, M., Kannt, A., Bamberg, E., Ludwig, B., Michel, H., and Fendler, K. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 4632-4636]. Here, we confirm that injection of the first electron into the fully oxidized cytochrome c oxidase from Paracoccus denitrificans is associated with a fast electrogenic 11 micros phase, but there is no further electrogenic phase up to 100 milliseconds when special care is taken to ensure that only fully oxidized enzyme is present initially. A slower electrogenic 135 micros phase only becomes apparent and grows in amplitude upon increasing the number of light flashes. This occurs in parallel with a decrease in amplitude of the 11 micros phase and correlates with the number of enzyme molecules that are already reduced by one electron before the flash. The electrogenic 135 micros phase does not appear with increasing flash number in the K354M mutant enzyme, where electron and proton transfer into the binuclear center is delayed. We conclude that the 135 micros phase, and its associated proton uptake, take place on electron injection into enzyme molecules where the binuclear heme a3-CuB site is already reduced by one electron, and that it is accompanied by oxidation of heme a with a similar time constant. Reduction of heme a is not associated with electrogenic proton uptake into the enzyme, neither in the fully oxidized nor in the one-electron-reduced enzyme. The extent of the electrogenic 135 micrcos phase also rules out the possibility that reduction of the binuclear center by the second electron would be coupled to proton translocation in addition to the electrogenic uptake of a proton.
通过对结合的钌[II]联吡啶进行激光闪光激发，将电子离散地注入脂质体中氧化型细胞色素c氧化酶，并通过时间分辨电位测定法记录膜电位。当一个电子从激发态染料经CuA中心转移到膜内相对介电深度d处的血红素a时，会在快速阶段产生膜电位[扎斯拉夫斯基，D.，考伦，A. D.，斯米尔诺娃，I. A.，维戈迪娜，T.，和康斯坦丁诺夫，A. A.（1993年）《欧洲生物化学学会联合会快报》336，389 - 393]。随后，膜电位可能在一个较慢的过程中进一步发展，这是由于质子从膜的另一侧转移到酶中[鲁伊滕贝格，M.，坎特，A.，班贝格，E.，路德维希，B.，米歇尔，H.，和芬德勒，K.（2000年）《美国国家科学院院刊》97，4632 - 4636]。在这里，我们证实，将第一个电子注入来自反硝化副球菌的完全氧化型细胞色素c氧化酶与一个快速的电生11微秒阶段相关，但在特别小心确保最初仅存在完全氧化型酶的情况下，直至100毫秒都没有进一步的电生阶段。一个较慢的电生135微秒阶段仅在增加闪光次数时才变得明显且幅度增大。这与11微秒阶段幅度的减小同时发生，并且与在闪光前已经被一个电子还原的酶分子数量相关。在K354M突变酶中，随着闪光次数增加，电生135微秒阶段不出现，在该突变酶中电子和质子向双核中心的转移被延迟。我们得出结论，135微秒阶段及其相关的质子摄取发生在电子注入到双核血红素a3 - CuB位点已经被一个电子还原的酶分子时，并且它伴随着血红素a以相似的时间常数被氧化。血红素a的还原与质子向酶中的电生性摄取无关，无论是在完全氧化型酶还是单电子还原型酶中。电生135微秒阶段的程度也排除了第二个电子对双核中心的还原除了质子的电生性摄取之外还会与质子转运偶联的可能性。
