Electron transfer reactions between Clostridium pasteurianum flavodoxin semiquinone and various oxidants (horse heart cytochrome c, ferricyanide, and ferric EDTA) have been studied as a function of ionic strength using stopped-flow spectrophotometry. The cytochrome c reaction is complicated by the existence of two cytochrome species which react at different rates and whose relative concentrations are ionic strength dependent. Only the faster of these two reactions is considered here. At low ionic strength, complex formation between cytochrome c and flavodoxin is indicated by a levelling-off of the pseudo-first order rate constant at high cytochrome c concentration. This is not observed for either ferricyanide or ferric EDTA. For cytochrome c, the rate and association constants for complex formation were found to increase with decreasing ionic strength, consistent with negative charges on flavodoxin interacting with the positively charged cytochrome electron transfer site. Both ferricyanide and ferric EDTA are negatively charged oxidants and the rate data respond to ionic strength changes as would be predicted for reactants of the same charge sign. These results demonstrate that electrostatic interactions involving negatively charged groups are important in orienting flavodoxin with respect to oxidants during electron transfer. The effects of structural modifications of the FMN prosthetic group of C. pasteurianum flavodoxin on the kinetics of electron transfer to the oxidized form (from 5-deazariboflavin semiquinone produced by laser flash photolysis) and from the semiquinone form (to horse heart cytochrome c using stopped-flow spectrophotometry) have been investigated. The analogs used were 7,8-dichloroFMN, 8-chloroFMN, 7-chloroFMN and 5,6,7,8-tetrahydroFMN. The ionic strength dependence of cytochrome c reduction was not affected by chlorine substitution, although the specific rate constants for complex formation and decay were appreciably smaller. On the other hand, all of the chlorine analogs had the same rate constant for deazariboflavin semiquinone oxidation. The rate constants for tetrahydroFMN flavodoxin semiquinone reduction of cytochrome c were considerably smaller than those for the native protein. The results for the chlorine analogs indicate the important roles that the polarity of the exposed flavin edge and the substitution of the 8 position play in electron transfer. The data obtained with the tetrahydroFMN analog indicates that the (pi) electron system of the flavin is necessary for rapid electron transfer. These implications are discussed for the electron transfer mechanism of flavodoxin.

The axial ligands of the iron porphyrin in Cytochrome c, an electron transfer protein, are an imidazole group of a histidine residue and a methionine thioether. This ligand coordination sphere has been difficult to model and consequently the influence of these ligands on the properties of cytochrome c has been problematic. The electrochemical and spectroscopic study of a novel strapped porphyrin has been addressed toward this problem. Spectroscopic studies have demonstrated the ability of this porphyrin to hold a thioether ligand near the central metal atom. The influence of the thioether is not seen in the UV/visible spectrum of the iron complex of this porphyrin. The coordination of N-methyl imidazole to the iron complexes of several porphyrins has been studied by UV/visible spectroscopy. These studies indicate a reduced affinity of the strapped porphyrin for this ligand. Also, the oxidation products of several porphyrins were monitored by thin-layer spectroelectrochemistry. Cyclic voltammetry has been used to demonstrate the influence of the thioether on the Fe('+3)/Fe('+2) electron transfer reaction. It was found that the thioether stabilizes the lower oxidation state causing an anodic shift in the half-wave potential for the reaction. However, the stabilization seen with this model system is not sufficient to account for the large positive redox potential of Cytochrome c. The oxidations of a selected group of free base and metallo- porphyrins were also studied. It was found that the oxidation of strapped porphyrins was similar in many respects to those of non-strapped porphyrins. The notable acception to this generalization was the instability of the cation radical of the strapped porphyrins used in this work.

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