title: 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.
abstract: 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.
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
authors.forename: R
authors.initials: R
authors.lastname: De Francesco
authors.forename: G
authors.initials: G
authors.lastname: Tollin
authors.forename: D E
authors.initials: DE
authors.lastname: Edmondson
mesh_terms: D003013:Clostridium / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism*; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology*; D005417:Flavobacterium / Q000378:metabolism; D005418:Flavodoxin / Q000378:metabolism*; D005420:Flavoproteins / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology*; D007202:Indicators and Reagents; D007700:Kinetics; D011809:Quinones; D013329:Structure-Activity Relationship
chemical_list: D003574:Cytochrome c Group; D005418:Flavodoxin; D005420:Flavoproteins; D007093:Imidazoles; D007202:Indicators and Reagents; D011809:Quinones; D005486:Flavin Mononucleotide