Brzezinski P, Malmström B G
Department of Biochemistry and Biophysics, Chalmers University of Technology, Göteborg, Sweden.
Biochim Biophys Acta. 1987 Oct 29;894(1):29-38. doi: 10.1016/0005-2728(87)90209-x.
Electron-transfer reactions following flash photolysis of the mixed-valence cytochrome oxidase-CO complex have been measured at 445, 598 and 830 nm between pH 5.2 and 9.0 in the temperature range of 0-25 degrees C. There is a rapid electron transfer from the cytochrome a3-CuB pair to CuA (time constant: 14200 s-1), which is followed by a slower electron transfer to cytochrome a. Both the rate and the amplitude of the rapid phase are independent of pH, and the rate in the direction from CuA to cytochrome a3-CuB is practically independent of temperature. The second phase depends strongly on pH due to the titration of an acid-base group with pKa = 7.6. The equilibrium at pH 7.4 corresponds to reduction potentials of 225 and 345 mV for cytochrome a and a3, respectively, from which it is concluded that the enzyme is in a different conformation compared to the fully oxidized form. The results have been used to suggest a series of reaction steps in a cycle of the oxidase as a proton pump. Application of the electron-transfer theory to the temperature-dependence data suggests a mechanism for electron gating in the pump. Reduction of both cytochrome a and CuA leads to a conformational change, which changes the structure of cytochrome a3-CuB in such a way that the reorganizational barrier for electron transfer is removed and the driving force is increased.
在0至25摄氏度的温度范围内，于pH 5.2至9.0之间，对混合价态的细胞色素氧化酶 - CO复合物进行闪光光解后的电子转移反应，在445、598和830纳米处进行了测量。存在从细胞色素a3 - CuB对向CuA的快速电子转移（时间常数：14200 s-1），随后是向细胞色素a的较慢电子转移。快速相的速率和幅度均与pH无关，并且从CuA到细胞色素a3 - CuB方向的速率实际上与温度无关。由于一个pKa = 7.6的酸碱基团的滴定，第二相强烈依赖于pH。在pH 7.4时的平衡对应于细胞色素a和a3的还原电位分别为225和345毫伏，由此得出该酶与完全氧化形式相比处于不同的构象。这些结果已被用于提出氧化酶作为质子泵的循环中的一系列反应步骤。将电子转移理论应用于温度依赖性数据表明了泵中电子门控的一种机制。细胞色素a和CuA的还原均导致构象变化，这种变化以这样一种方式改变细胞色素a3 - CuB的结构，即消除了电子转移的重组障碍并增加了驱动力。
