Caffrey M S, Bartsch R G, Cusanovich M A
Department of Biochemistry, University of Arizona, Tucson 85721.
J Biol Chem. 1992 Mar 25;267(9):6317-21.
Photooxidation of Rhodobacter capsulatus cytochrome c2 and four site-directed mutants by detergent solubilized Rhodobacter sphaeroides reaction centers was studied as a function of ionic strength at pH 8.0. Mutants of cytochrome c2 included K12D (lysine 12 substituted by aspartate), K14E (lysine 14 substituted by glutamate), K32E (lysine 32 substituted by glutamate), and K14E/K32E (lysines 14 and 32 substituted by glutamates). With respect to the wild-type, the mutants exhibited decreased second-order rate constants, indicating perturbation of their electrostatic interaction with the reaction center. In the transient complex, the interaction domain charges of the reaction center and wild-type cytochrome c2 were estimated to be -4.8 and +4.8, respectively. In contrast, the interaction domain charges of mutants K12D, K14E, K32E, and K14E/K32E were estimated to be +2.8, +3.7, +3.6 and +1.3, respectively. At infinite ionic strength, the second-order rate constant of the wild-type cytochrome c2 photooxidation (k infinity) was estimated to be 8.7 x 10(6) M-1 s-1. In the case of K32E, k infinity was not changed significantly (8.2 x 10(6) m-1 s-1), suggesting that the electrostatic perturbation of this mutant was largely overcome at high ionic strength. In contrast, the k infinity for K12D, K14E, and K14E/K32E were estimated to be decreased 2-7-fold. Consequently, mutations to R. capsulatus lysines 12 and 14 appear to perturb the distance and/or orientation of the cytochrome c2 relative to the reaction center in the reactive complex, as well as alter electrostatic interactions. Based upon the kinetic results presented here, the cytochrome c2-reaction center transient complex has been modeled.
研究了在pH 8.0条件下，离子强度对荚膜红细菌细胞色素c2及其四个定点突变体被去污剂溶解的球形红细菌反应中心光氧化的影响。细胞色素c2的突变体包括K12D（赖氨酸12被天冬氨酸取代）、K14E（赖氨酸14被谷氨酸取代）、K32E（赖氨酸32被谷氨酸取代）和K14E/K32E（赖氨酸14和32被谷氨酸取代）。与野生型相比，突变体的二级速率常数降低，表明其与反应中心的静电相互作用受到干扰。在瞬态复合物中，反应中心与野生型细胞色素c2的相互作用域电荷估计分别为-4.8和+4.8。相比之下，突变体K12D、K14E、K32E和K14E/K32E的相互作用域电荷估计分别为+2.8、+3.7、+3.6和+1.3。在无限离子强度下，野生型细胞色素c2光氧化的二级速率常数（k∞）估计为8.7×10⁶ M⁻¹ s⁻¹。对于K32E，k∞没有显著变化（8.2×10⁶ M⁻¹ s⁻¹），这表明该突变体的静电干扰在高离子强度下基本被克服。相比之下，K12D、K14E和K14E/K32E的k∞估计降低了2至7倍。因此，荚膜红细菌赖氨酸12和14的突变似乎扰乱了反应复合物中细胞色素c2相对于反应中心的距离和/或方向，同时也改变了静电相互作用。基于此处给出的动力学结果，对细胞色素c2 - 反应中心瞬态复合物进行了建模。
