Kinetic studies of reduction of a 1:1 cytochrome c-flavodoxin complex by free flavin semiquinones and rubredoxin.
Kinetic studies of reduction of a 1:1 cytochrome c-flavodoxin complex by free flavin semiquinones and rubredoxin.
复制标题
游离黄素半醌和红氧还蛋白还原 1:1 细胞色素 c-黄素还蛋白复合物的动力学研究。
DOI：
10.1021/bi00359a035
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Tollin,G
中科院分区：
文献类型：
--
作者：
Hazzard,JT;Cusanovich,MA;Tainer,JA;Getzoff,ED;Tollin,G
Revised Manuscript Received January 14, 1986 abstract: 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 therate 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 neutralflavin 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 C12FMN 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 dueto uncompensated flavodoxin carboxylates, as predicted by Matthew et al.[Matthew, JB, 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-orderelectron-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 havebeen 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. e nature of the intermediate complex formed by a pair of electron-transfer proteins during their reaction is of interest in understanding the factors that govern the interaction mechanisms of physiological redoxcouples. Since direct structure determinations of such complexes have not thus far been achieved, computer models of several electron-transfer complexes have been generated on the basis of crystallographic structures of the isolated proteins (Salemme, 1976; Poulos & Kraut, 1980; Simondsen et al., 1982; Poulos & Mauk, 1983). The unifying themes underlying such models of electrontransfer complexes (Salemme, 1978) are that the protein-protein interaction is primarily electrostatic in nature, the prosthetic groups are located at the protein interface in a relatively nonpolar environment (ie, H20 is excluded from the interaction domain), and the prosthetic groups are oriented with their planes relatively parallel to one another and are close enough such that direct electron …
