Hazzard J T, Cusanovich M A, Tainer J A, Getzoff E D, Tollin G
Biochemistry. 1986 Jun 3;25(11):3318-28. doi: 10.1021/bi00359a035.
The kinetics of reduction by free flavin semiquinones and reduced rubredoxin of the individual components of the 1:1 complex formed between horse heart cytochrome c and Clostridium pasteurianum flavodoxin have been studied. Complex formation did not affect the rate constant for reduction of flavodoxin by 5-deazariboflavin semiquinone, indicating that the accessibility of the flavin mononucleotide (FMN) of complexed flavodoxin is the same as in the free protein. Reduction of the complexed cytochrome c by the neutral flavin semiquinones of lumiflavin and riboflavin was significantly affected by complex formation (2-3-fold rate constant decrease), indicating that there are steric constraints on the accessibility of the cytochrome heme to small exogenous reductants. Reduction of complexed cytochrome c by the negatively charged semiquinones of FMN and Cl2FMN was also characterized. A repulsive electrostatic interaction between the reductants and complexed cytochrome was observed, whereas with free cytochrome an attractive interaction had previously been found. This is consistent with the presence of negative electrostatic potential at the protein interface due to uncompensated flavodoxin carboxylates, as predicted by Matthew et al. [Matthew, J. B., Weber, P. C., Salemme, F. R., & Richards, F. M. (1983) Nature (London) 301, 169-171]. Further, pseudo-first-order rate constants for the reduction of complexed cytochrome by these flavins had a nonlinear concentration dependence, rather than obeying simple second-order kinetics. This is interpreted by using a mechanism involving a rate-determining structural isomerization of the protein complex prior to the second-order electron-transfer step. The magnitude of the decrease in the rate constant for reduction of complexed cytochrome c by the negatively charged reduced rubredoxin was approximately the same as observed for free flavins. Furthermore, simple second-order kinetics were obtained, and the apparent electrostatic interaction between rubredoxin and the complex was attractive. These results suggest that flavodoxin was partially displaced from its complex with cytochrome c by a collisional interaction with rubredoxin. The effects of complexation on the kinetics have been correlated with a solvent-accessible surface representation of the computer-generated model of the flavodoxin-cytochrome c complex [Simondsen, R. P., Weber, P. C., Salemme, F. R., & Tollin, G. (1982) Biochemistry 21, 6366-6375]. The experimental observations are generally consistent with the structural model but clearly require the invocation of dynamic motions at the protein-protein interface.
已对马心细胞色素c与巴氏梭菌黄素氧还蛋白形成的1:1复合物中各组分被游离黄素半醌和还原型铁氧化还原蛋白还原的动力学进行了研究。复合物的形成不影响5-脱氮核黄素半醌对黄素氧还蛋白的还原速率常数，这表明复合黄素氧还蛋白中黄素单核苷酸（FMN）的可及性与游离蛋白中的相同。复合物中的细胞色素c被黄素和核黄素的中性黄素半醌还原受到复合物形成的显著影响（速率常数降低2至3倍），这表明细胞色素血红素对外源小还原剂的可及性存在空间限制。还对FMN和Cl2FMN的带负电荷的半醌对复合细胞色素c的还原进行了表征。观察到还原剂与复合细胞色素之间存在排斥性静电相互作用，而对于游离细胞色素，此前发现的是吸引性相互作用。这与Matthew等人预测的由于未补偿的黄素氧还蛋白羧酸盐在蛋白质界面处存在负静电势一致[Matthew, J. B., Weber, P. C., Salemme, F. R., & Richards, F. M. (1983) Nature (London) 301, 169 - 171]。此外，这些黄素对复合细胞色素还原的准一级速率常数具有非线性浓度依赖性，而不是遵循简单的二级动力学。这通过一种机制来解释，该机制涉及在二级电子转移步骤之前蛋白质复合物的速率决定结构异构化。带负电荷的还原型铁氧化还原蛋白对复合细胞色素c还原的速率常数降低幅度与游离黄素观察到的大致相同。此外，得到了简单的二级动力学，并且铁氧化还原蛋白与复合物之间的表观静电相互作用是吸引性的。这些结果表明，黄素氧还蛋白通过与铁氧化还原蛋白的碰撞相互作用部分地从其与细胞色素c的复合物中被取代。复合物形成对动力学的影响已与黄素氧还蛋白 - 细胞色素c复合物计算机生成模型的溶剂可及表面表示相关联[Simondsen, R. P., Weber, P. C., Salemme, F. R., & Tollin, G. (1982) Biochemistry 21, 6366 - 6375]。实验观察结果总体上与结构模型一致，但显然需要考虑蛋白质 - 蛋白质界面处的动态运动。
