Brändén Gisela, Pawate Ashtamurthy S, Gennis Robert B, Brzezinski Peter
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Proc Natl Acad Sci U S A. 2006 Jan 10;103(2):317-22. doi: 10.1073/pnas.0507734103. Epub 2006 Jan 3.
Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain and couples energetically the reduction of oxygen to water to proton pumping across the membrane. The results from previous studies showed that proton pumping can be uncoupled from the O2-reduction reaction by replacement of one single residue, Asn-139 by Asp (N139D), located approximately 30 A from the catalytic site, in the D-proton pathway. The uncoupling was correlated with an increase in the pK(a) of an internal proton donor, Glu-286, from approximately 9.4 to >11. Here, we show that replacement of the acidic residue, Asp-132 by Asn in the N139D CcO (D132N/N139D double-mutant CcO) results in restoration of the Glu-286 pK(a) to the original value and recoupling of the proton pump during steady-state turnover. Furthermore, a kinetic investigation of the specific reaction steps in the D132N/N139D double-mutant CcO showed that proton pumping is sustained even if proton uptake from solution, through the D-pathway, is slowed. However, during single-turnover oxidation of the fully reduced CcO the P --> F transition, which does not involve electron transfer to the catalytic site, was not coupled to proton pumping. The results provide insights into the mechanism of proton pumping by CcO and the structural elements involved in this process.
细胞色素c氧化酶（CcO）是呼吸链的末端酶，它能将氧气还原为水的过程与跨膜质子泵的能量耦合。先前的研究结果表明，通过将位于D质子途径中距催化位点约30埃处的单个残基天冬酰胺-139替换为天冬氨酸（N139D），质子泵可与O2还原反应解偶联。这种解偶联与内部质子供体谷氨酸-286的pK(a)从约9.4增加到>11相关。在此，我们表明在N139D CcO中（D132N/N139D双突变CcO）将酸性残基天冬氨酸-132替换为天冬酰胺会导致谷氨酸-286的pK(a)恢复到原始值，并在稳态周转期间使质子泵重新偶联。此外，对D132N/N139D双突变CcO中特定反应步骤的动力学研究表明，即使通过D途径从溶液中摄取质子的速度减慢，质子泵仍能持续。然而，在完全还原的CcO的单周转氧化过程中，不涉及向催化位点电子转移的P→F转变与质子泵不偶联。这些结果为CcO质子泵的机制以及该过程中涉及的结构元件提供了见解。
