Complex formation between flavodoxin and cytochrome c. Cross-linking studies.

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.

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confidence: 99%

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

Palma

Moura

LeGall³

et al. 1994

“…In some cases, covalent cross-linking was used. This latter methodology has been applied to two electron transfer complexes, horse cytochrome c-Azotobacter vinelandii flavodoxin (Dickerson et al, 1985) and horse cytochrome c-yeast cytochrome c peroxidase (Pettigrew & Seilman, 1982; Waldmeyer et al, 1982;Waldmeyer & Bosshard, 1985;Bechtold & Bosshard, 1985). In the latter complex, a 16-fold decrease 1 Abbreviations: cytochrome c(III) and c(II), ferric and ferro cytochrome c, respectively; CcP(III) and CcP(II), ferric and ferro cytochrome c peroxidase, respectively; CcP(IV,R,+), peroxidase species oxidized by H202 yielding an Fe(IV) and an oxidized amino acid, R"+ (i.e., compound I); CcP(IV), peroxidase species oxidized to the Fe(IV) state without R group oxidation; EDTA, ethylenediaminetetraacetic acid; LFH", RFH", FMNH', and 5-DRFH', neutral semiquinone species of lumiflavin, riboflavin, flavin mononucleotide, and 5-deazariboflavin, respectively; NADP+, oxidized nicotinamide adenine dinucleotide phosphate; £m,7, midpoint reduction potential measured at pH 7. in the rate constant for ascorbate reduction of complexed cytochrome c was observed, as well as a 95% decrease in peroxidase activity toward exogenous cytochrome c(II).…”

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confidence: 99%

Kinetics of reduction by free flavin semiquinones of the components of the cytochrome c-cytochrome c peroxidase complex and intracomplex electron transfer

Hazzard¹,

Poulos²,

Tollin³

1987

101

The kinetics of reduction by free flavin semiquinones of the individual components of 1:1 complexes of yeast ferric and ferryl cytochrome c peroxidase and the cytochromes c of horse, tuna, and yeast (iso-2) have been studied. Complex formation decreases the rate constant for reduction of ferric peroxidase by 44%. On the basis of a computer model of the complex structure [Poulos, T.L., & Finzel, B.C. (1984) Pept. Protein Rev. 4, 115-171], this decrease cannot be accounted for by steric effects and suggests a decrease in the dynamic motions of the peroxidase at the peroxide access channel caused by complexation. The orientations of the three cytochromes within the complex are not equivalent. This is shown by differential decreases in the rate constants for reduction by neutral flavin semiquinones upon complexation, which are in the order tuna much greater than horse greater than yeast iso-2. Further support for differences in orientation is provided by the observation that, with the negatively charged reductant FMNH., the electrostatic environments near the horse and tuna cytochrome c electron-transfer sites within their respective complexes with peroxidase are of opposite sign. For the horse and tuna cytochrome c complexes, we have also observed nonlinear concentration dependencies of the reduction rate constants with FMNH.. This is interpreted in terms of dynamic motion at the protein-protein interface. We have directly measured the physiologically significant intra-complex one electron transfer rate constants from the three ferrous cytochromes c to the peroxide-oxidized species of the peroxidase. At low ionic strength these rate constants are 920, 730, and 150 s-1 for tuna, horse, and yeast cytochromes c, respectively. These results are also consistent with the contention that the orientations of the three cytochromes within the complex with CcP are not the same. The effect on the intracomplex electron-transfer rate constant of the peroxidase amino acid side chain(s) that is (are) oxidized by the reduction of peroxide was determined to be relatively small. Thus, the rate constant for reduction by horse cytochrome c of the peroxidase species in which only the heme iron atom is oxidized was decreased by only 38%, indicating that this oxidized side-chain group is not tightly coupled to the ferryl peroxidase heme iron. Finally, it was found that, in the absence of cytochrome c, neither of the ferryl peroxidase species could be rapidly reduced by flavin semiquinones.(ABSTRACT TRUNCATED AT 400 WORDS)

“…A previous comparative study (Cheddar et al, 1986) of Clostridium pasteurianum and Azotobacter vinelandii flavodoxins has shown the latter to be considerably less reactive than the former, despite the significantly lower redox potential for the oxidized/semiquinone couple of the Azotobacter protein, which would provide a larger thermodynamic driving force resulting in an increased rate of electron transfer. Cross-linking studies have shown that Azotobacter flavodoxin forms a tight 1:1 complex with cytochrome c, with an estimated association constant of 4 X 104 M"1 at 88 mM ionic strength (Dickerson et al, 1985), which compares favorably with the kinetically determined value for cytochrome c and C. pasteurianum flavodoxin semiquinone (18 X 104 M"1) ( Simondsen et al, 1982). More recent studies in this laboratory have shown that the kinetically determined Kz for Clostridium flavodoxin is approximately the same as that for Azotobacter flavodoxin (De Francesco et al, 1977), whereas the rate constant for electron transfer is approximately 4-fold greater for Clostridium than for Azotobacter flavodoxin.…”

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confidence: 99%

Flavodoxin-cytochrome c interactions: circular dichroism and nuclear magnetic resonance studies

Tollin¹,

Brown²,

Francesco³

et al. 1987

Circular dichroism and 1H and 31P nuclear magnetic resonance spectroscopy have been used to investigate complex formation between cytochrome c and the flavodoxins from Azotobacter vinelandii and Clostridium pasteurianum. Such complexes are known to be involved in the mechanism of electron transfer between these two redox proteins. A large increase in ellipticity in the Soret band of the cytochrome heme was observed upon formation of the Clostridium flavodoxin complex, whereas much smaller changes were found for the complexes with either Azotobacter flavodoxin or an 8 alpha-imidazolyl-FMN-substituted Clostridium flavodoxin analogue. Similarly, the magnitudes of the perturbations of the contact-shifted heme proton resonances obtained upon complexation of cytochrome c by Azotobacter flavodoxin were much smaller than those previously shown for Clostridium flavodoxin [Hazzard, J. T., & Tollin, G. (1985) Biochem. Biophys. Res. Commun. 130, 1281-1286]. 31P nuclear magnetic resonance measurements were also consistent with differences in the interactions between the components in the complexes of the two flavodoxins with cytochrome c. It is suggested that these spectral changes are due to a loosening or opening of the heme crevice upon Clostridium flavodoxin binding, which allows closer contact between the heme and flavin prosthetic groups and results in a faster rate of electron transfer. The implications of these observations for biological oxidation-reduction processes are considered.