Evaluation of the role of specific acidic amino acid residues in electron transfer between the flavodoxin and cytochrome c3 from Desulfovibrio vulgaris.

Abstract

A hypothetical model for electron transfer complex between cytochrome c3 and the flavodoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris has been proposed, based on electrostatic potential field calculations and NMR data [Stewart, D. E., LeGall, J. , Moura, I., Moura, J. J. G., Peck, H. D., Jr., Xavier, A. V., Weiner, P. K., & Wampler, J. E. (1988) Biochemistry 27, 2444-2450]. This modeled complex relies primarily on the formation of five ion pairs between lysine residues of the cytochrome and acidic residues surrounding the flavin mononucleotide cofactor of the flavodoxin. In this study, the role of several acidic residues of the flavodoxin in the formation of this complex and in electron transfer between these two proteins was evaluated. A total of 17 flavodoxin mutants were studied in which 10 acidic amino acids--Asp62, Asp63, Glu66, Asp69, Asp70, Asp95, Glu99, Asp106, Asp127, and Asp129--had been permanently neutralized either individually or in various combinations by substitution with their amide amino acid equivalent (i.e., asparate to asparagine, glutamate to glutamine) through site-directed mutagenesis. The kinetic data for the transfer of electrons from reduced cytochrome c3 to the various flavodoxin mutants do not conform well to a simple bimolecular mechanism involving the formation of an intermediate electron transfer complex. Instead, a minimal electron transfer mechanism is proposed in which an initial complex is formed that is stabilized by intermolecular electrostatic interactions but is relatively inefficient in terms of electron transfer. This step is followed by a rate-limiting reorganization of that complex leading to efficient electron transfer. The apparent rate of this reorganization step was enhanced by the disruption of the initial electrostatic interactions through the neutralization of certain acidic amino acid residues leading to faster overall observed electron transfer rates at low ionic strengths. Of the five acidic residues involved in ion pairing in the modeled complex proposed by Stewart et al. (1988), the kinetic data strongly implicate Asp62, Glu66, and Asp95 in the formation of the electrostatic interactions that control electron transfer. Less certainty is provided by this study for the involvement of Asp69 and Asp129, although the data do not exclude their participation. It was not possible to determine whether the modeled complex represents the optimal configuration for electron transfer obtained after the reorganization step or actually represents the initial complex. The data do provide evidence for the importance of electrostatic interactions in electron transfer between these two proteins and for the existence of alternative binding modes involving acidic residues on the surface of the flavodoxin other than those proposed in that model.