期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：11
页码：3397-3402
DOI：10.1073/pnas.1422434112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceCytochrome c oxidase is the terminal electron acceptor in mitochondria and aerobic bacteria, where O2 reduction is linked to proton pumping across the membrane. Understanding the mechanism by which the enzyme pumps protons requires identification of the so-called proton-loading site, which is the controlling element that assures unidirectionality in the proton flux. The position of this site has been predicted on the basis of theoretical calculations but has not been identified in experimental studies. Here we have used sophisticated biophysical techniques to investigate intraprotein electron and proton transfer in the thermophilic bacterium Thermus thermophilus. The data made it possible to identify the destination of the pumped proton and to unravel the sequence of reactions that leads to proton translocation. The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba3 oxidase where a putative "pump site" was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O2 reduction. The results from our studies show that proton uptake to the pump site (time constant [~]65 s in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of [~]1.2 ms was slowed to [~]8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba3 cytochrome c oxidase.
关键词：cytochrome c oxidase ; membrane protein ; respiration ; cytochrome aa 3 ; electron transfer
