Influence of 8 alpha-imidazole substitution of the FMN cofactor on the rate of electron transfer from the neutral semiquinones of two flavodoxins to cytochrome c.

The effects of substituting an imidazole ring onto the 8 alpha-position of the FMN cofactor on the kinetics of electron transfer from the neutral semiquinone forms of Azotobacter and Clostridium flavodoxins to oxidized horse heart cytochrome c have been investigated by stopped-flow methods. Although 8 alpha-substitution does not alter the mechanistic pathway of the reaction, the rate constants are decreased by factors of 10-30, without significant changes in the equilibrium association constants of the intermediate electron-transfer complexes. Protonation of the imidazole ring further decreases the observed second-order rate constants for the electron-transfer reaction by factors of 20-50. The pKa values for the 8 alpha-imidazole ring in both flavodoxin semiquinones were determined to be approximately 7. In contrast, the reactions of the native flavodoxins with cytochrome c are pH independent. The results are consistent with a structural model of the intermediate complex [Simondsen, R. P., Weber, P. C., Salemme, F. R., & Tollin, G. (1982) Biochemistry 21, 6366-6375], which postulates a close fit between the exposed dimethylbenzene ring of the FMN and the heme edge within a nonpolar interface region. The results further indicate that electron transfer is uncoupled from proton transfer, that it is the rate-limiting step, and that it occurs prior to proton transfer at all pH values. Finally, the results do not provide support for a direct role of the imidazole ring in the facilitation of one-electron transfer in those enzymes containing 8 alpha-N-histidylflavin coenzymes.