Evidence for a Ternary Complex Formed between Flavodoxin and Cytochrome c3: 1H-NMR and Molecular Modeling Studies

Abstract

Small electron-transfer proteins such as flavodoxin (16 kDa) and the tetraheme cytochrome C-3 (13 kDa) have been used to mimic, in vitro, part of the complex electron-transfer chain operating between substrate electron donors and respiratory electron accepters, in sulfate-reducing bacteria (Desulfovibrio species). The nature and properties of the complex formed between these proteins are revealed by H-1-NMR and molecular modeling approaches. Our previous study with the Desulfovibrio vulgaris proteins [Moura, I., Moura, J. J. G., Santos; M. H., & Xavier, A. V. (1980) Cienc. Biol. (Portugal) 5, 195-197; 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] indicated that the complex between cytochrome c(3) and flavodoxin could be monitored by changes in the NMR signals of the heme methyl groups of the cytochrome and that the electrostatic surface charge (Coulomb's law) on the two proteins favored interaction between one unique heme of the cytochrome with flavodoxin. If the interaction is indeed driven by the electrostatic complementarity between the acidic flavodoxin and a unique positive region of the cytochrome c(3) other homologous proteins from these two families of proteins might be expected to interact similarly. In this study, three homologous Desulfovibrio cytochromes c(3) were used. which show a remarkable variation in their individual isoelectric points (ranging from 5.5 to 9.5). On the basis of data obtained from protein-protein titrations followed at specific proton NMR signals (i.e., heme methyl resonances), a binding model for this complex has been developed with evaluation of stoichiometry and binding constants. This binding model involves one site on the cytochromes c(3) and two sites on the flavodoxin, with formation oa ternary complex at saturation. In order to understand the potential chemical form of the binding model, a structural model for the hypothetical ternary complex, formed between one molecule of Desulfovibrio salexigens flavodoxin and two molecules of cytochrome c(3), is proposed. These molecular models of the complexes were constructed on the basis of complementarity of Coulombic electrostatic surface potentials, using the available X-ray structures of the isolated proteins and, when required, model structures (D. salexigens flavodoxin and Desulfovibrio desulfuricans ATCC 27774 cytochrome c(3)) predicted by homology modeling.