PROTON-PUMPING BY CYTOCHROME-OXIDASE AS STUDIED BY TIME-RESOLVED STOPPED-FLOW SPECTROPHOTOMETRY

被引：24
作者：
ANTONINI, G
MALATESTA, F
SARTI, P
BRUNORI, M
机构：
[1] UNIV ROMA LA SAPIENZA,DEPT BIOCHEM SCI,PIAZZALE ALDO MORO 5,I-00185 ROME,ITALY
[2] UNIV ROMA TOR VERGATA,DEPT EXPTL MED & BIOCHEM SCI,ROME,ITALY
[3] UNIV ROMA LA SAPIENZA,CONSIGLIO NAZL RIC CTR MOLEC BIOL,I-00185 ROME,ITALY
[4] UNIV CAGLIARI,INST BIOL CHEM,I-09100 CAGLIARI,ITALY
来源：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 1993年 / 90卷 / 13期
关键词：
D O I：
10.1073/pnas.90.13.5949
中图分类号：
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号：
07 ; 0710 ; 09 ;
摘要：
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).
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页码：5949 / 5953
页数：5
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