EVALUATION OF THE ROLE OF SPECIFIC ACIDIC AMINO-ACID-RESIDUES IN ELECTRON-TRANSFER BETWEEN THE FLAVODOXIN AND CYTOCHROME C(3) FROM DESULFOVIBRIO-VULGARIS [HILDENBOROUGH]

A hypothetical model for electron transfer complex between cytochrome cs and the flavodoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris has been proposed, based on electrostatic potential field ca lculations 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 o f the cytochrome and acidic residues surrounding the flavin mononucleo tide cofactor of the flavodoxin. In this study, the role of several ac idic residues of the flavodoxin in the formation of this complex and i n electron transfer between these two proteins was evaluated. A total of 17 flavodoxin mutants were studied in which 10 acidic amino acids-A sp62, Asp63, Glu66, Asp69, Asp70, Asp95, Glu99, Asp106, Asp127, and As p129-had been permanently neutralizedeither individually or in various combinations by substitution with their amide amino acid equivalent ( i.e., asparate to asparagine, glutamate to glutamine) through site-dir ected mutagenesis. The kinetic data for the transfer of electrons from reduced cytochrome cs to the various flavodoxin mutants do not confor m well to a simple bimolecular mechanism involving the formation of an intermediate electron transfer complex. Instead, a minimal electron t ransfer mechanism is proposed in which an initial complex is formed th at is stabilized by intermolecular electrostatic interactions but is r elatively inefficient in terms of electron transfer. This step is foll owed by a rate-limiting reorganization of that complex leading to effi cient electron transfer. The apparent rate of this reorganization step was enhanced by the disruption of the initial electrostatic interacti ons through the neutralization of certain acidic amino acid residues l eading 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 s trongly implicate Asp62, Glu66, and Asp95 in the formation of the elec trostatic interactions that control electron transfer. Less certainty is provided by this study for the involvement of Asp69 and Asp129, alt hough the data do not exclude their participation. It was not possible to determine whether the modeled complex represents the optimal confi guration for electron transfer obtained after the reorganization step or actually represents the initial complex. The data do provide eviden ce for the importance of electrostatic interactions in electron transf er between these two proteins and for the existence of alternative bin ding modes involving acidic residues on the surface of the flavodoxin other than those proposed in that model.