Lepp Håkan, Salomonsson Lina, Zhu Jia-Peng, Gennis Robert B, Brzezinski Peter
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Biochim Biophys Acta. 2008 Jul-Aug;1777(7-8):897-903. doi: 10.1016/j.bbabio.2008.04.013. Epub 2008 Apr 16.
Cytochrome c oxidase is a membrane-bound enzyme, which catalyses the one-electron oxidation of four molecules of cytochrome c and the four-electron reduction of O(2) to water. Electron transfer through the enzyme is coupled to proton pumping across the membrane. Protons that are pumped as well as those that are used for O(2) reduction are transferred though a specific intraprotein (D) pathway. Results from earlier studies have shown that replacement of residue Asn139 by an Asp, at the beginning of the D pathway, results in blocking proton pumping without slowing uptake of substrate protons used for O(2) reduction. Furthermore, introduction of the acidic residue results in an increase of the apparent pK(a) of E286, an internal proton donor to the catalytic site, from 9.4 to ~11. In this study we have investigated intramolecular electron and proton transfer in a mutant cytochrome c oxidase in which a neutral residue, Thr, was introduced at the 139 site. The mutation results in uncoupling of proton pumping from O(2) reduction, but a decrease in the apparent pK(a) of E286 from 9.4 to 7.6. The data provide insights into the mechanism by which cytochrome c oxidase pumps protons and the structural elements involved in this process.
细胞色素c氧化酶是一种膜结合酶，它催化四个细胞色素c分子的单电子氧化以及将O₂四电子还原为水。通过该酶的电子传递与跨膜质子泵浦相偶联。被泵浦的质子以及用于O₂还原的质子通过特定的蛋白质内（D）途径转移。早期研究结果表明，在D途径起始处将Asn139残基替换为Asp，会导致质子泵浦受阻，而不会减慢用于O₂还原的底物质子的摄取。此外，引入酸性残基会导致E286（催化位点的内部质子供体）的表观pK(a)从9.4增加到约11。在本研究中，我们研究了一种突变型细胞色素c氧化酶中的分子内电子和质子转移，该突变型在139位点引入了一个中性残基Thr。该突变导致质子泵浦与O₂还原解偶联，但E286的表观pK(a)从9.4降至7.6。这些数据为细胞色素c氧化酶泵浦质子的机制以及该过程中涉及的结构元件提供了见解。
