Antonini G, Malatesta F, Sarti P, Brunori M
Department of Experimental Medicine and Biochemical Sciences, University of Rome, Tor Vergata, Italy.
J Biol Chem. 1991 Jul 15;266(20):13193-202.
The kinetics of cytochrome oxidase reconstituted into small phospholipid vesicles (COV) has been followed by transient optical spectroscopy under steady-state and pre-steady-state conditions, in the presence and absence of ionophores. The effect of valinomycin on the activity of reconstituted cytochrome oxidase is shown to depend on the absolute concentration of the ionophore and on the number of turnovers elapsed by the enzyme; this novel observation, which escaped previous investigations, may account for important differences in results and therefore in interpretation of the mechanism of control of the enzyme activity as between Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), Gregory and Ferguson-Miller (Gregory, L., and Ferguson-Miller, S. (1989) Biochemistry 28, 2655-2662) and Capitanio et al. (Capitanio, N., De Nitto, E., Villani, G., Capitanio, G., and Papa, S. (1990) Biochemistry 29, 2939-2944). Quantitative analysis of the optical spectra acquired within 10 ms over a large wavelength and time range (500-650 nm and 5 ms to 60 s) under different experimental conditions, indicates that the electrical component of the transmembrane electrochemical gradient controls the rate of the internal electron transfer from cytochrome a-CuA to cytochrome a3-CuB as well as the cytochrome c to cytochrome a electron transfer. The slow down of cytochrome oxidase activity observed in the presence of valinomycin after several (greater than 10) turnovers is attributed to alkalinization of the vesicle interior, which affects the internal electron transfer rate. These two mechanisms of control act most likely independently. A "cubic scheme," which illustrates the effect of the electrochemical gradient on two states of cytochrome oxidase characterized by different redox and proton pumping activities is presented and discussed.
已通过瞬态光谱法在稳态和预稳态条件下，在有离子载体和无离子载体的情况下，跟踪了重组到小磷脂囊泡（COV）中的细胞色素氧化酶的动力学。结果表明，缬氨霉素对重组细胞色素氧化酶活性的影响取决于离子载体的绝对浓度以及酶经历的周转次数；这一先前研究未发现的新观察结果，可能解释了结果以及对酶活性控制机制解释方面的重要差异，这些差异存在于布鲁诺里等人（Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365 - 2368）、格雷戈里和弗格森 - 米勒（Gregory, L., and Ferguson - Miller, S. (1989) Biochemistry 28, 2655 - 2662）以及卡皮塔尼奥等人（Capitanio, N., De Nitto, E., Villani, G., Capitanio, G., and Papa, S. (1990) Biochemistry 29, 2939 - 2944）的研究之间。在不同实验条件下，对在10毫秒内、大波长和时间范围（500 - 650纳米和5毫秒至60秒）获取的光谱进行定量分析表明，跨膜电化学梯度的电成分控制着从细胞色素a - CuA到细胞色素a3 - CuB的内部电子转移速率以及细胞色素c到细胞色素a的电子转移速率。在经过数次（大于10次）周转后，在缬氨霉素存在下观察到的细胞色素氧化酶活性下降归因于囊泡内部的碱化，这影响了内部电子转移速率。这两种控制机制很可能独立起作用。本文提出并讨论了一个“立方图”，该图说明了电化学梯度对细胞色素氧化酶两种具有不同氧化还原和质子泵浦活性状态的影响。
