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 c3 (13 kDa) have been used to mimic, in vitro, part of the complex electron-transfer chain operating between substrate electron donors and respiratory electron acceptors, in sulfate-reducing bacteria (Desulfovibrio species). The nature and properties of the complex formed between these proteins are revealed by 1H-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 c3 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 c3, other homologous proteins from these two families of proteins might be expected to interact similarly. In this study, three homologous Desulfovibrio cytochromes c3 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 c3 and two sites on the flavodoxin, with formation of a 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 c3, 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 c3) predicted by homology modeling.

NMR characterization and solution structure determination of the oxidized cytochrome c 7 from Desulfuromonas acetoxidans

“…A short right-handed 3 10 helix (His-22-Glu-26), four segments of right-handed ␣-helix (Glu-29-His-34, Ser-63-Ala-70, Ser-78-Lys-90, and Leu-93-Gly-99), and a short doublestranded antiparallel ␤-sheet (Val-9-Gly-13 and Lys-16-Phe-20) are observed in the former structure. Despite the low sequence homology found among various Cyt c 3 , the x-ray structures up to now reported reveal highly conserved secondary and tertiary structure elements (60-90%) (3)(4)(5)(6)(7)37), where always a ␤-sheet and four helices are present. The main tertiary structural feature of the known Cyt c 3 is the substantially unchanged relative spatial arrangement of the four heme moieties.…”

The Ternary Complex of Cytochrome f and Cytochrome c: Identification of a Second Binding Site and Competition for Plastocyanin Binding

“…A binding model with two cytc molecules per cytf resulted in a greatly improved fit. The two-site model for nonequivalent, noninteracting sites [21,22] (solid line) yielded binding constants of approximately 2 Â 10 4 M À1 and 4 Â 10 3 M À1 (Table 1).…”

Negatively Charged Anabaena Flavodoxin Residues (Asp144 and Glu145) Are Important for Reconstitution of Cytochrome P450 17α-Hydroxylase Activity

“…Most conserved residues in alignments between Flds and P450 reductase, for instance, lie within regions that bind FMN. In addition, clusters of negatively charged residues exist in these proteins, which have been proposed to be involved in the recognition of specific positively charged residues on their corresponding redox partners (11)(12)(13)(14).…”