Kinetic studies of reduction of a 1:1 cytochrome c-flavodoxin complex by free flavin semiquinones and rubredoxin.
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.
D003013:Clostridium / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism*; D005288:Ferredoxins / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism*; D005418:Flavodoxin / Q000378:metabolism*; D005420:Flavoproteins / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012416:Rubredoxins / Q000378:metabolism*
D003574:Cytochrome c Group; D005288:Ferredoxins; D005415:Flavins; D005418:Flavodoxin; D005420:Flavoproteins; D012416:Rubredoxins; D005486:Flavin Mononucleotide