EVIDENCE FOR A TERNARY COMPLEX FORMED BETWEEN FLAVODOXIN AND CYTOCHROME C(3) - H-1-NMR AND MOLECULAR MODELING STUDIES

Small electron-transfer proteins such as flavodoxin (16 kDa) and the t etraheme cytochrome C-3 (13 kDa) have been used to mimic, in vitro, pa rt of the complex electron-transfer chain operating between substrate electron donors and respiratory electron accepters, in sulfate-reducin g bacteria (Desulfovibrio species). The nature and properties of the c omplex formed between these proteins are revealed by H-1-NMR and molec ular modeling approaches. Our previous study with the Desulfovibrio vu lgaris proteins [Moura, I., Moura, J. J. G., Santos; M. H., & Xavier, A. V. (1980) Cienc. Biol. (Portugal) 5, 195-197; Stewart, D, E., LeGal l, J., Moura, I., Moura, J. J. G., Peck, H. D., Jr., Xavier, A. V., We iner, P. K., & Wampler, J. E. (1988) Biochemistry 27, 2444-2450] indic ated that the complex between cytochrome c(3) and flavodoxin could be monitored by changes in the NMR signals of the heme methyl groups of t he cytochrome and that the electrostatic surface charge (Coulomb's law ) on the two proteins favored interaction between one unique heme of t he 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 protein s from these two families of proteins might be expected to interact si milarly. In this study, three homologous Desulfovibrio cytochromes c(3 ) were used. which show a remarkable variation in their individual iso electric points (ranging from 5.5 to 9.5). On the basis of data obtain ed from protein-protein titrations followed at specific proton NMR sig nals (i.e., heme methyl resonances), a binding model for this complex has been developed with evaluation of stoichiometry and binding consta nts. This binding model involves one site on the cytochromes c(3) and two sites on the flavodoxin, with formation oa ternary complex at satu ration. In order to understand the potential chemical form of the bind ing model, a structural model for the hypothetical ternary complex, fo rmed between one molecule of Desulfovibrio salexigens flavodoxin and t wo molecules of cytochrome c(3), is proposed. These molecular models o f the complexes were constructed on the basis of complementarity of Co ulombic electrostatic surface potentials, using the available X-ray st ructures of the isolated proteins and, when required, model structures (D. salexigens flavodoxin and Desulfovibrio desulfuricans ATCC 27774 cytochrome c(3)) predicted by homology modeling.