Rosen P, Pecht I
Biochemistry. 1976 Feb 24;15(4):775-86. doi: 10.1021/bi00649a008.
The redox reaction between cytochrome c (Cyt c) (P-551) and the blue copper protein azurin, both from Pseudomonas aeruginosa, was studied using the temperature-jump technique. Two relaxation times were observed in a mechanism assumed to involve three equilibria. The fast relaxation time (0.4 less than tau less than 8 ms) was ascribed to the electron exchange step. The slow relaxation time (tau congruent to 37 ms) was assigned to a conformational equilibrium of the reduced azurin that was coupled through the electron exchange step to a faster conformational equilibrium of the oxidized Cyt c (P551). But because the Cyt c (P551) isomerization, being very rapid, was uncoupled from the two slower equilibria, and was assumed to involve no spectral change, the amplitude of its relaxation time (tau congruent to 0.1 ms) would be zero. At 25 degrees C and pH 7.0 the rate constants for the oxidation and reduction of Cyt c (P551) by azurin were 6.1 X 10(6) and 7.8 X 10(6) M-1 s-1, respectively; for the formation and disappearance of the reactive conformational isomer of azurin they were 12 and 17 s-1, respectively. The rates for the Cyt c (P551) isomerization could only be estimated at approximately 10(4) s-1. The thermodynamic parameters of each reaction step were evaluated from the amplitudes of the relaxations and from Eyring plots of the rate constants. Measurements of the overall equilibrium constant showed it to be temperature independent (5-35 degrees C), i.e. deltaHtot = 0. This zero enthalpy change was found to be compatible with the enthalpies calculated for the individual steps. In the electron exchange equilibrium, the values of the activation enthalpies were two to three times higher than the values published for various low molecular weight reagents in their electron exchange with copper proteins, yet the rate of exchange between Cyt c (P551) and azurin was some hundreds of times faster. This was explained in terms of the measured positive or zero entropies of activation that could result from a high level of specificity between the proteins particularly in areas of complementary charges. The mechanism of electron transfer was considered as essentially an outer sphere reaction, of which the rate could be approximated by the Marcus theory.
利用温度跃变技术研究了来自铜绿假单胞菌的细胞色素c（Cyt c）（P-551）与蓝色铜蛋白天青蛋白之间的氧化还原反应。在假定涉及三个平衡的机制中观察到两个弛豫时间。快速弛豫时间（0.4＜τ＜8毫秒）归因于电子交换步骤。缓慢弛豫时间（τ≈37毫秒）归因于还原型天青蛋白的构象平衡，该平衡通过电子交换步骤与氧化型Cyt c（P551）的更快构象平衡相耦合。但由于Cyt c（P551）的异构化非常迅速，与两个较慢的平衡解耦，且假定不涉及光谱变化，其弛豫时间（τ≈0.1毫秒）的幅度将为零。在25℃和pH 7.0时，天青蛋白氧化和还原Cyt c（P551）的速率常数分别为6.1×10⁶和7.8×10⁶ M⁻¹ s⁻¹；天青蛋白反应性构象异构体的形成和消失速率常数分别为12和17 s⁻¹。Cyt c（P551）异构化的速率只能估计约为10⁴ s⁻¹。从弛豫幅度和速率常数的艾林图评估了每个反应步骤的热力学参数。对总平衡常数的测量表明其与温度无关（5-35℃），即ΔHtot = 0。发现该零焓变与为各个步骤计算的焓值相符。在电子交换平衡中，活化焓的值比各种低分子量试剂与铜蛋白进行电子交换时公布的值高两到三倍，但Cyt c（P551）与天青蛋白之间的交换速率快数百倍。这可以用测量到的正或零活化熵来解释，这可能是由于蛋白质之间特别是在互补电荷区域的高度特异性所致。电子转移机制被认为基本上是一个外层球反应，其速率可以用马库斯理论近似。
