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The gene encoding Desulfovibrio (D.) vulgaris rubrerythrin (Prickril, B. C., Kurtz, D. M., Jr., LeGall, J., & Voordouw, G. (1991) Biochemistry 30, 1118), a protein of unknown function containing both FeS4 and (mu-oxo)diiron sites, was cloned and overexpressed in Escherichia coli. Upon cell lysis, the overexpressed protein was found in an insoluble form deficient in iron. Iron was incorporated in vitro by dissolving the protein in 3 M guanidinium chloride, adding Fe(II) anaerobically and diluting the denaturant. This recombinant rubrerythrin was found to have properties very similar to those of rubrerythrin isolated from D. vulgaris, except that the recombinant rubrerythrin contained six rather than four (or five) iron atoms per 44 kDa homodimer. Analyses of UV-vis, Mössbauer, and EPR spectra showed that the six iron atoms in recombinant rubrerythrin are organized as two FeS4 and two (mu-oxo/hydroxo)diiron sites. In order to allow examination of the diiron sites in the absence of the FeS4 sites, a truncated gene encoding the N-terminal 152 residues of D. vulgaris rubrerythrin was also cloned and overexpressed as an insoluble protein in E. coli, and iron was incorporated by a procedure analogous to that for recombinant rubrerythrin. This so-called "chopped" rubrerythrin (CRr) was found to consist of an approximately 35 kDa homodimer containing four iron atoms. Spectroscopic characterization indicated that the four iron atoms in CRr are organized as two diiron sites, the majority of which closely resemble the (mu-oxo)diiron(III) sites in E. coli ribonucleotide reductase R2 protein, and a minor fraction of which resemble the mixed-valent diiron(II,III) site in methane monooxygenase hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)

Desulfoferrodoxin, a non-heme iron protein, was purified previously from extracts of Desulfovibrio desulfuricans (ATCC 27774) (Moura, I., Tavares, P., Moura, J. J. G., Ravi, N., Huynh, B. H., Liu, M.-Y., and LeGall, J. (1990) J. Biol. Chem. 265, 21596-21602). The as-isolated protein displays a pink color (pink form) and contains two mononuclear iron sites in different oxidation states: a ferric site (center I) with a distorted tetrahedral sulfur coordination similar to that found in desulforedoxin from Desulfovibrio gigas and a ferrous site (center II) octahedrally coordinated with predominantly nitrogen/oxygen-containing ligands. A new form of desulfoferrodoxin which displays a gray color (gray form) has now been purified. Optical, electron paramagnetic resonance (EPR), and Mössbauer data of the gray desulfoferrodoxin indicate that both iron centers are in the high-spin ferric states. In addition to the EPR signals originating from center I at g = 7.7, 5.7, 4.1, and 1.8, the gray form of desulfoferrodoxin exhibits a signal at g = 4.3 and a shoulder at g = 9.6, indicating a high-spin ferric state with E/D approximately 1/3 for the oxidized center II. Redox titrations of the gray form of the protein monitored by optical spectroscopy indicate midpoint potentials of +4 +/- 10 and +240 +/- 10 mV for centers I and II, respectively. Mössbauer spectra of the gray form of the protein are consistent with the EPR finding that both centers are high-spin ferric and can be analyzed in terms of the EPR-determined spin Hamiltonian parameters. The Mössbauer parameters for both the ferric and ferrous forms of center II are indicative of a mononuclear high spin iron site with octahedral coordination and predominantly nitrogen/oxygen-containing ligands. Resonance Raman studies confirm the structural similarity of center I and the distorted tetrahedral FeS4 center in desulforedoxin and provide evidence for one or two cysteinyl-S ligands for center II. On the basis of the resonance Raman results, the 635 nm absorption band that is responsible for the gray color of the oxidized protein is assigned to a cysteinyl-S-->Fe(III) charge transfer transition localized on center II. The novel properties and possible function of center II are discussed in relation to those of mononuclear iron centers in other enzymes.

Rubrerythrin, a contraction of rubredoxin and hemerythrin, is the trivial name given to a non-heme iron protein isolated from Desulfovibrio vulgaris (Hildenborough). This protein, whose physiological function is unknown, was first characterized by J. LeGall et al. [(1988) Biochemistry 28, 1636] as being a homodimer of subunit M(r) = 21,900 with four Fe per homodimer distributed as two rubredoxin-type FeS4 centers and one hemerythrin-type diiron cluster. Subsequent analysis of the amino acid sequence of the rubrerythrin gene [Kurtz, D. M., Jr., & Prickril, B.C. (1991) Biochem. Biophys. Res. Commun. 181, 137] revealed an internal homology which suggested that each subunit can accommodate one diiron cluster. Here, we report a procedure for reconstitution of the as-isolated D. vulgaris rubrerythrin with 57Fe. The reconstituted protein was characterized by optical, electron paramagnetic resonance, and Mössbauer spectroscopies. The results indicate successful incorporation of 57Fe into the two types of sites and strongly suggest that each subunit of rubrerythrin can indeed accommodate one diiron cluster as well as one rubredoxin-type center. Combined with amino acid sequence analysis, the spectroscopic characterization further suggests that the rubrerythrin subunit contains a diiron site whose structure is more closely related to that in ribonucleotide reductase than to that in hemerythrin.

A new type of non-heme iron protein was purified to homogeneity from extracts of Desulfovibrio desulfuricans (ATCC 27774) and Desulfovibrio vulgaris (strain Hildenborough). This protein is a monomer of 16-kDa containing two iron atoms per molecule. The visible spectrum has maxima at 495, 368, and 279 nm and the EPR spectrum of the native form shows resonances at g = 7.7, 5.7, 4.1 and 1.8 characteristic of a high-spin ferric ion (S = 5/2) with E/D = 0.08. Mössbauer data indicates the presence of two types of iron: an FeS4 site very similar to that found in desulforedoxin from Desulfovibrio gigas and an octahedral coordinated high-spin ferrous site most probably with nitrogen/oxygen-containing ligands. Due to this rather unusual combination of active centers, this novel protein is named desulfoferrodoxin. Based on NH2-terminal amino acid sequence determined so far, the desulfoferrodoxin isolated from D. desulfuricans (ATCC 27774) appears to be a close analogue to a recently discovered gene product from D. vulgaris (Brumlik, M.J., and Voordouw, G. (1989) J. Bacteriol. 171, 49996-50004), which was suggested to be a rubredoxin oxidoreductase. However, reduced pyridine nucleotides failed to reduce the desulforedoxin-like center of this new protein.

The tetrameric form of a Desulfovibrio gigas ferredoxin, named Fd II, mediates electron transfer between cytochrome c3 and sulfite reductase. We have studied two stable oxidation states of this protein with Mössbauer spectroscopy and electron paramagnetic resonance. We found 3 iron atoms/monomer and a spin concentration of 0.9 spins/monomer for the oxidized protein. Taken together, the EPR and Mössbauer data demonstrate conclusively the presence of a spin-coupled structure containing 3 iron atoms and labile sulfur. The Mössbauer data show also that this metal center is structurally similar, if not identical, with the low potential center of a ferredoxin from Azotobacter vinelandii, a novel cluster described recently (Emptage, M.H., Kent, T.A., Huynh, B.H., Rawlings, J., Orme-Johnson, W.H., and Münck, E. (1980) J. Biol. Chem. 255, 1793-1796).

Recent Mössbauer and EPR studies of two ferredoxins and of aconitase have given evidence for a three-iron cluster, probably of a [3Fe-3S] type. The studies of the oxidized EPR-active centers have shown that the three iron sites are characterized by significantly different magnetic hyperfine coupling constants. For the ferredoxin from Azotobacter vinelandii, for instance, we have observed A1 = -41 MHz, A2 = +18 MHz, and [A3] = 5 MHz. We demonstrate here that the magnetic properties of the clusters can be explained with a simple model of three high-spin ferric ions (S = 5/2) exchange-coupled to a system spin S = 1/2. The model assumes isotropic exchange and different couplings between the iron sites. The results show that the three sites have intrinsic hyperfine interactions similar to those of ferric rubredoxin; the differences in the observed interactions reflect the geometrical features of spin coupling. Furthermore, the three exchange coupling constants are equal within a factor of 2. This implies that the three-iron cluster is a single covalently linked structure and should not be considered as a [2Fe-2S] cluster weakly coupled to a third iron atom.

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)

Methanosarcina barkeri ferredoxin was purified and characterized by electron paramagnetic resonance (EPR) and Mössbauer spectroscopy. The purification procedure included chromatographic steps on DEAE-cellulose and gel filtration. The isolated protein is unstable under aerobic conditions. The ferredoxin exhibits charge transfer bands at 283 nm and 405 nm with an absorption ratio A405/A283 = 0.73. Its molecular weight has been estimated to be 20000-22000 by gel filtration chromatography. The native ferredoxin exhibits an intense EPR signal at g = 2.02 and only a very weak g = 1.94 signal develops upon reduction with dithionite. The Mössbauer spectra of the reduced protein are characteristic of a [3Fe-3S] center. The combined EPR and Mössbauer studies show that M. barkeri ferredoxin contains only [3Fe-3S] clusters, similar to Azotobacter vinelandii Fd[Emptage, M.H., Kent, T.A., Huynh, B.H., Rawlings, J., Orme-Johnson, W.H. & Münck, M. (1980) J. Biol. Chem. 255, 1793-1796], Desulfovibrio gigas FdII [Huynh, B.H., Moura, J.J.G., Moura, I., Kent, T.A., LeGall, J., Xavier, A.V. & Münck, E. (1980) J. Biol. Chem. 255, 3242-3244] and mitochondrial beef heart aconitase [Kent, T.A., Dreyer, J.-L., Kennedy, M.C., Huynh, B.H., Emptage, M.H., Beinert, H. & Münck, E. (1982) Proc. Natl Acad. Sci. USA, 79, 1096-1100].

The soluble ferredoxin from Thermus thermophilus was examined by Mössbauer and EPR spectroscopies and by reductive titrations. These studies demonstrate the presence of one 3Fe center, responsible for the characteristic g = 2.02 EPR signal in the oxidized protein, and one [4Fe-4S] center which is responsible for the rhombic EPR spectrum of the fully reduced protein. These assignments should replace those made by Ohnishi et al. (Ohnishi, T., Blum, H., Sato, S., Nakazawa, K., Hon-nami, K., and Oshima, T. (1980) J. Biol. Chem. 255, 345-348) prior to the discovery of the 3Fe clusters. The amino acid composition was determined and is discussed with reference to recent structural studies of 7Fe ferredoxins.