Antonini G, Malatesta F, Sarti P, Brunori M
Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Italy.
Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):5949-53. doi: 10.1073/pnas.90.13.5949.
The H+/e- stoichiometry for the proton pump of cytochrome c oxidase reportedly varies between 0 and 1, depending on experimental conditions. In this paper, we report the results obtained by a combination of transient optical spectroscopy with a time resolution of 10 ms and a singular value decomposition analysis to follow the kinetics, separate the observed spectral components, and quantitate the stoichiometry of the pump. By using cytochrome oxidase reconstituted into small unilamellar vesicles, we show that the time courses of ferrocytochrome c oxidation and phenol red acidification or alkalinization fit a simple kinetic scheme. The fitting procedure leads to unbiased and objective determination of the H+/e- ratio under various experimental conditions. The proton-pumping stoichiometry was found to be 1.01 +/- 0.10, independent of the number of turnovers, proton back-leak rate, or type of experiment (oxidant or reductant pulse).
据报道，细胞色素c氧化酶质子泵的H⁺/e⁻化学计量比在0到1之间变化，这取决于实验条件。在本文中，我们报告了通过结合时间分辨率为10毫秒的瞬态光学光谱和奇异值分解分析所获得的结果，以跟踪动力学、分离观察到的光谱成分并定量泵的化学计量比。通过使用重构到小单层囊泡中的细胞色素氧化酶，我们表明亚铁细胞色素c氧化和酚红酸化或碱化的时间进程符合一个简单的动力学方案。拟合过程能够在各种实验条件下无偏差且客观地确定H⁺/e⁻比值。发现质子泵化学计量比为1.01±0.10，与周转数、质子反向泄漏率或实验类型（氧化剂或还原剂脉冲）无关。
