Jorge Lampreia

{Dodecaheme cytochrome c has been purified from Desulfovibrio (D.) desulfuricans ATCC 27774 cells grown under both nitrate and sulfate-respiring conditions. Therefore, it is likely to play a role in the electron-transfer system of both respiratory chains. Its molecular mass (37 768 kDa) was determined by electrospray mass spectrometry. Its first 39 amino acids were sequenced and a motif was found between amino acids 32 and 37 that seems to exist in all the cytochromes of the c3 type from sulfate-reducing bacteria sequenced at present. The midpoint redox potentials of this cytochrome were estimated to be -68, -120, -248 and -310 mV. Electron paramagnetic resonance spectroscopy of the oxidized cytochrome shows several low-spin components with a g(max) spreading from 3.254 to 2.983. Two crystalline forms were obtained by vapour diffusion from a solution containing 2% PEG 6000 and 0.25-0.75 M acetate buffer pH = 5.5. Both crystals belong to monoclinic space groups: one is PZ,, with a = 61.00

Flavodoxin was isolated and purified from Desulfovibrio desulfuricans ATCC 27774, a sulfatereducing organism that can also utilize nitrate as an alternative electron acceptor. Mid-point oxidation-reduction potentials of this flavodoxin were determined by ultraviolet/visible and EPR methods coupled to potentiometric measurements and their pH dependence studied in detail. The redox potential E(2), for the couple oxidized/semiquinone forms at pH 6.7 and 25 degrees C is -40 mV, while the value for the semiquinone/hydroquinone forms (E(1)), at the same pH, -387 mV. E(2) varies linearly with pH, while E(1) is independent of pH at high values. However, at low pH (<7.0), this value is less negative, compatible with a redox-linked protonation of the flavodoxin hydroquinone. A comparative study is presented for Desulfovibrio salexigens NCIB 8403 flavodoxin {[}Moura, I., Moura, J. J. G., Bruschi, M. and LeGall, J. (1980) Biochim. Biophys. Acta 591, 1-8]. The complete primary amino acid sequence was obtained by automated Edman degradation from peptides obtained by chemical and enzymic procedures. The amino acid sequence was confirmed by FAB/MS. Using the previously determined tridimensional structure of Desulfovibrio vulgaris flavodoxin as a model {[}similarity, 48,6%; Watenpaugh, K. D., Sieker, L. C., Jensen, L. H., LeGall, J. and Dubourdieu M. (1972) Proc. Natl Acad. Sci. USA 69, 3185-3188], the tridimensional structure of D. desulfuricans ATCC 27774 flavodoxin was predicted using AMBER force-field calculations.

The primary structure of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774, a redox protein with two mononuclear iron sites, was determined by automatic Edman degradation and mass spectrometry of the composing peptides, It contains 125 amino acid residues of which five are cysteines, The first four, Cys-9, Cys-12, Cys-28 and Cys-29, are responsible for the binding of Center I which has a distorted tetrahedral sulfur coordination similar to that found in desulforedoxin from D. gigas, The remaining Cys-115 is proposed to be involved in the coordination of Center II, which is probably octahedrally coordinated with predominantly nitrogen/oxygen containing ligands as previously suggested by Mossbauer and Raman spectroscopy.