Pn. Palma et al., EVIDENCE FOR A TERNARY COMPLEX FORMED BETWEEN FLAVODOXIN AND CYTOCHROME C(3) - H-1-NMR AND MOLECULAR MODELING STUDIES, Biochemistry, 33(21), 1994, pp. 6394-6407
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.