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.

The crystal structure of desulforedoxin from Desulfovibrio gigas, a new homo-dimeric (2x36 amino acids) non-heme iron protein, has been solved by the SIRAS method using the indium-substituted protein as the single derivative. The structure was refined to a crystallographic X-factor of 16.9% at 1.8 Angstrom resolution. Native desulforedoxin crystals were grown from either PEG 4K or lithium sulfate, with cell constants a = b = 42.18 Angstrom, = 72.22 Angstrom (for crystals grown from PEG 4K), and they belong to space group P3(2)21. The indium-substituted protein crystallized isomorphously under the same conditions. The 2-fold symmetric dimer is firmly hydrogen bonded and folds as an incomplete beta-barrel with the two iron centers placed on opposite poles of the molecule. Each iron atom is coordinated to four cysteinyl residues in a distorted tetrahedral arrangement. Both iron atoms are 16 Angstrom apart but connected across the 2-fold axis by 14 covalent bonds along the polypeptide chain plus two hydrogen bonds. Desulforedoxin and rubredoxin share some structural features but show significant differences in terms of metal environment and water structure, which account for the known spectroscopic differences between rubredoxin and desulforedoxin. (C) 1995 Academic Press Limited

The dsr gene from Desulfovibrio gigas encoding the nonheme iron protein desulforedoxin was cloned using the polymerase chain reaction, expressed in Escherichia coli, and purified to homogeneity. The physical and spectroscopic properties of the recombinant protein resemble those observed for the native protein isolated from D. gigas. These include an alpha(2) tertiary structure, the presence of bound iron, and absorbance maxima at 370 and 506 nm in the UV/visible spectrum due to ligand-to-iron charge transfer bands. Low temperature electron paramagnetic resonance studies confirm the presence of a high spin ferric ion with g values of 7.7, 5.7, 4.1, and 1.8. Interestingly, E. coli produced two forms of desulforedoxin containing iron. One form was identified as a dimer with the metal-binding sites of both subunits occupied by iron while the second form contained equivalent amounts of iron and zinc and represents a dimer with one subunit occupied by iron and the second with zinc.

Desulforedoxin is a protein purified from cellular extracts of Desulfovibrio gigas. It is a small (7.9 kDa) dimeric protein that contains a distorted rubredoxin like center (one single iron coordinated by four cysteinyl residues). Due to the simplicity of the polypeptide chain and of the iron center, an attempt was made to chemically produce this protein. A 36 amino acid polypeptide chain was synthesized based on the known sequence of native Desulforedoxin. The iron center was then reconstituted and the biochemical and spectroscopic characteristics of this synthetic protein were investigated. The final product has an equal sequence to the protein purified from D. gigas. The synthetic and natural Dr are very similar, in terms of redox potential and spectroscopic properties (UV-Visible, EPR, Mossbauer). (C) 1995 Academic Press, Inc.