A new non-heme iron protein from the periplasmic fraction of Desulfovibrio vulgaris (Hildenbourough NCIB 8303) has been purified to homogeneity, and its amino acid composition, molecular weight, redox potential, iron content, and optical, EPR, and Mössbauer spectroscopic properties have been determined. This new protein is composed of two identical subunits with subunit molecular weight of 21,900 and contains four iron atoms per molecule. The as-purified oxidized protein exhibits an optical spectrum with absorption maxima at 492, 365, and 280 nm, and its EPR spectrum shows resonances at g = 4.3 and 9.4, characteristic of oxidized rubredoxin. The Mössbauer data indicate the presence of approximately equal amounts of two types of iron; we named them the Rd-like and the Hr-like iron due to their similarity to the iron centers of rubredoxins (Rds) and hemerythrins (Hrs), respectively. For the Rd-like iron, the measured fine and hyperfine parameters (D = 1.5 cm-1, E/D = 0.26, delta EQ = -0.55 mm/s, delta = 0.27 mm/s, Axx/gn beta n = -16.5 T, Ayy/gn beta n = -15.6 T, and Azz/gn beta n = -17.0 T) are almost identical with those obtained for the rubredoxin from Clostridium pasteurianum. Redox-titration studies monitored by EPR, however, showed that these Rd-like centers have a midpoint redox potential of +230 +/- 10 mV, approximately 250 mV more positive than those reported for rubredoxins. Another unusual feature of this protein is the presence of the Hr-like iron atoms.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

Ni and Se x-ray absorption spectroscopic studies of the [NiFeSeihydrogenases from Desulfovibrio baculatus are described. The Ni site geometry is pseudo-octahedral with a coordinating ligand composition of 3-4 (N,O) at 2.06 A, 1-2 (S,CI) at 2.17 A, and 1 Se at 2.44 A. The Se coordination environment consists of 1 C at 2.0 A and a heavy scatterer M (M = Ni or Fe) at -2.4 A. These results are interpreted in terms of a selenocysteine residue coordinated to the Ni site. The possible role of the Ni-Se site in the catalytic activation of H2 is discussed.Hydrogenases are vital to the anaerobic metabolism of sulfate-reducing bacteria and many other types of bacteria. Hydrogenases catalyze the bidirectional activation of molecular hydrogen H2 = 2H+ + 2e-and their activities are routinely determined by H2 production, H2 utilization, or the 2H2-H+ exchange assays (1). In the past, it was generally accepted that a single hydrogenase carried out these simple redox reactions (1), but in recent years the unveiling of the structural diversity of hydrogenases has promoted the idea that different hydrogenases may reflect differences in cellular localization and metabolic function (2). This specificity suggests that structural differences are the basis for tailoring the hydrogenase reaction to meet metabolic demands. The elucidation of the structural details of the active sites of hydrogenases is the first step toward a molecular understanding of the mechanisms involved in the hydrogenase reaction.In the genus Desulfovibrio, the metabolism of hydrogen involves at least three types of hydrogenases that may be distinguished by their heavy element composition, immunological reactivities, and gene structures (3-6). The [Fe]hydrogenases contain only iron-sulfur clusters (7-9), the [NiFe] As part of our effort to elucidate the structures of the nickel sites in the Ni-containing hydrogenases, and to define a structural basis for the functional differences between the Seand the non-Se-containing hydrogenases (19, 20), we report here the results of Ni and Se x-ray absorption spectroscopic measurements on the [NiFeSe]hydrogenases ofD. baculatus. MATERIALS AND METHODS Sample Preparation. The D. baculatus [NiFeSe]hydrogenasesample was prepared according to the procedure described in ref. 19. To obtain a sample -2 mM Ni and Se, the periplasmic, cytoplasmic, and membrane-bound fractions were combined. Total iron was determined by the 2,4,6-tripyridyl-1,3,5-triazine method (21), and metals were quantified by plasma emission spectroscopy using a Mark II Jarrell-Ash model 965 AtomComp (Fisher). Nickel was also determined by atomic absorption spectroscopy. The sample for x-ray absorption spectroscopy

No abstract

The gene coding for rubrerythrin from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) has been cloned and sequenced. Rubrerythrin is known to contain two types of iron sites: one rubredoxin-like FeS4 center in each of the two identical subunits and one hemerythrin-like diiron site per dimer [LeGall, J., et al. (1988) Biochemistry 27, 1636-1642]. The gene encodes a polypeptide of 191 amino acids, and a normal ribosome binding site is located 11-6 base pairs upstream from the translational start of the gene. There is no evidence for the presence of a leader sequence, suggesting a cytoplasmic location for the protein. The rubrerythrin gene is not part of any other known transcriptional unit in the D. vulgaris genome. The nucleotide sequence encodes four Cys residues, the minimum required for ligation to iron in rubredoxin. The pairs of Cys residues occur in Cys-X-X-Cys sequences as they do in rubredoxin, but the 12-residue spacing between the Cys pairs in rubrerythrin is less than half that in rubredoxins. A pair of Arg residues flanking one Cys residue may contribute to the much more positive reduction potential of the rubredoxin-like site in rubrerythrin compared to that of rubredoxin. While the amino acid sequence of rubrerythrin shows no significant overall homology with that of any known protein, the C-terminal region does share some homology with rubredoxin sequences. If folding of the rubredoxin-like amino acid sequence domain in rubrerythrin is similar to that in rubredoxins, then three His residues are brought into proximity.(ABSTRACT TRUNCATED AT 250 WORDS)