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 Mossbauer 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 Mossbauer data demonstrate conclusively the presence of a spin-coupled structure containing 3 iron atoms and labile sulfur. The Mossbauer 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 Munck, E. (1980) J. Biol. Chem. 255, 1793-1796).

Mössbauer, EPR, and biochemical techniques were used to characterize two dissimilatory sulfite reductases: desulforubidin from Desulfovibrio baculatus strain DSM 1743 and desulfoviridin from Desulfovibrio gigas . For each molecule of desulforubidin, there are two sirohemes and four [4Fe−4S] clusters. The [4Fe−4S] clusters are in the diamagnetic 2+ oxidation state. The sirohemes are high-spin ferric (S=5/2) and each siroheme is exchanged-coupled to a [4Fe−4S] 2+ cluster. Such an exchange-coupled siroheme-[4Fe−4S] unit has also been found in the assimilatory sulfite reductase from Escherichia coli /1/ and in a low-molecular weight sulfite reductase from Desulfovibrio vulgaris /2/. For each molecule of defulfoviridin, there are two tetrahydroporphyrin groups and four [4Fe−4S] 2+ clusters. To our surprise, we discovered that about 80% of the tetrahydroporphyrin groups, however, do not bind iron.

The [NiFe] hydrogenase isolated from Desulfovibrio gigas was poised at different redox potentials and studied by Mossbauer spectroscopy. The data firmly establish that this hydrogenase contains four prosthetic groups: one nickel center, one [3Fe-xS], and two [4Fe-4S] clusters. In the native enzyme, both the nickel and the [3Fe-xS] cluster are EPR-active. At low temperature (4.2 K), the [3Fe-xS] cluster exhibits a paramagnetic Mossbauer spectrum typical for oxidized [3Fe-xS] clusters. At higher temperatures (greater than 20 K), the paramagnetic spectrum collapses into a quadrupole doublet with parameters magnitude of delta EQ magnitude of = 0.7 +/- 0.06 mm/s and delta = 0.36 +/- 0.06 mm/s, typical of high-spin Fe(III). The observed isomer shift is slightly larger than those observed for the three-iron clusters in D. gigas ferredoxin II (Huynh, B. H., Moura, J. J. G., Moura, I., Kent, T. A., LeGall, J., Xavier, A. V., and Munck, E. (1980) J. Biol. Chem. 255, 3242-3244) and in Azotobacter vinelandii ferredoxin I (Emptage, M. H., Kent, T. A., Huynh, B. H., Rawlings, J., Orme-Johnson, W. H., and Munck, E. (1980) J. Biol. Chem. 255, 1793-1796) and may indicate a different iron coordination environment. When D. gigas hydrogenase is poised at potentials lower than -80 mV (versus normal hydrogen electrode), the [3Fe-xS] cluster is reduced and becomes EPR-silent. The Mossbauer data indicate that the reduced [3Fe-xS] cluster remains intact, i.e. it does not interconvert into a [4Fe-4S] cluster. Also, the electronic properties of the reduced [3Fe-xS] cluster suggest that it is magnetically isolated from the other paramagnetic centers.

The resonance Raman spectra of ferredoxins (Fd) I and II from Desulfovibrio gigas are reported using 4579 A Ar+ laser excitation. The (3Fe-3S) center in Fd II has a characteristic resonance Raman spectrum, readily distinguishable from those of (2Fe-2S) or (4Fe-4S) clusters. Reduction of Fd II produces a marked alteration in the resonance Raman spectrum. Fd I is shown to contain both (3Fe-3S) and (4Fe-4S) Fd-type clusters. The results illustrate the potential of resonance Raman spectroscopy in Fe-S cluster identification, even in cases where more than one cluster type is present.

Quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni (QH-EDH) contains two cofactors, 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinoline-4,5-dione (PQQ) and heme c. Since previous studies on the kinetics of this enzyme suggested that both participate in electron transfer, spectroscopic investigations were performed of the oxidized and reduced holo- and apoenzyme (without PQQ but with heme c) to reveal the nature of the interaction between the two redox centers. From this it appears that the properties of the heme in the enzyme are affected by the presence of PQQ, as judged from the shift of the maxima in the ultraviolet/visible absorption spectra of the heme moiety in both reduced and oxidized QH-EDH and the 60-mV increase of the heme midpoint redox potential caused by PQQ addition. Also 1H-NMR spectroscopy was indicative for interaction since binding of PQQ induced shifts in the resonances of the methyl groups of the porphyrin ring in the oxidized form of the apoenzyme and a shift in the methionine heme ligand resonance of the reduced form of the apoenzyme. On the other hand, resonance Raman spectra of the heme in the different enzyme forms were nearly similar. These results suggest that a major effect of PQQ binding to apo-QH-EDH is a rotation of the methionine ligand of heme c. Since no intermediate 1H-NMR spectra were observed upon titration of apoenzyme with PQQ, apparently no exchange occurs of PQQ between (oxidized) holo- and apoenzyme at the NMR time scale and at that of the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Treponema pallidum, the causative agent of venereal syphilis, is a microaerophilic obligate pathogen of humans. As it disseminates hematogenously and invades a wide range of tissues, T. pallidum presumably must tolerate substantial oxidative stress. Analysis of the T. pallidum genome indicates that the syphilis spirochete lacks most of the iron-binding proteins present in many other bacterial pathogens, including the oxidative defense enzymes superoxide dismutase, catalase, and peroxidase, but does possess an orthologue (TP0823) for neelaredoxin, an enzyme of hyperthermophilic and sulfate-reducing anaerobes shown to possess superoxide reductase activity. To analyze the potential role of neelaredoxin in treponemal oxidative defense, we examined the biochemical, spectroscopic, and antioxidant properties of recombinant T. pallidum neelaredoxin. Neelaredoxin was shown to be expressed in T. pallidum by reverse transcriptase-polymerase chain reaction and Western blot analysis. Recombinant neelaredoxin is a 26-kDa alpha(2) homodimer containing, on average, 0.7 iron atoms/subunit. Mossbauer and EPR analysis of the purified protein indicates that the iron atom exists as a mononuclear center in a mixture of high spin ferrous and ferric oxidation states. The fully oxidized form, obtained by the addition of K(3)(Fe(CN)(6)), exhibits an optical spectrum with absorbances at 280, 320, and 656 nm; the last feature is responsible for the protein's blue color, which disappears upon ascorbate reduction. The fully oxidized protein has a A(280)/A(656) ratio of 10.3. Enzymatic studies revealed that T. pallidum neelaredoxin is able to catalyze a redox equilibrium between superoxide and hydrogen peroxide, a result consistent with it being a superoxide reductase. This finding, the first description of a T. pallidum iron-binding protein, indicates that the syphilis spirochete copes with oxidative stress via a primitive mechanism, which, thus far, has not been described in pathogenic bacteria.

Desulforedoxin and the N-terminus of desulfoferrodoxin share a 36 amino acid domain containing a (Cys-S)(4) metal binding site. Recombinant forms of desulforedoxin, an N-terminal fragment of desulfoferrodoxin, and two desulforedoxin mutant proteins were reconstituted with Fe3+ Cd2+, and Zn2+ and relative metal ion affinities assessed by proton titrations. Protons compete with metal for protein ligands, a process that can be followed by monitoring the optical spectrum of the metal-protein complex as a function of pH. For all polypeptides, Fe3+ bound with the highest affinity, whereas the affinity of Zn2+ was greater than Cd2+ in desulforedoxin and the N-terminal fragment of desulfoferrodoxin, but this order was reversed in desulforedoxin mutant proteins. Metal binding in both mutants was significantly impaired. Furthermore, the Fe3+ complex of both mutants underwent a time-dependent bleaching process which coincided with increased reactivity of cysteine residues to Ellman's reagent and concomitant metal dissociation. It is hypothesized that this results from an autoredox reaction in which Fe3+ is reduced to Fe2+ with attendant oxidation of ligand thiols.

Adenylate kinase (AK; ATP:AMP phosphotransferase; EC 2.7.4.3) is involved in the reversible transfer of the terminal phosphate group from ATP to AMP. AKs contribute to the maintenance of a constant level of cellular adenine nucleotides, which is necessary for the energetic metabolism of the cell. Three metal ions, cobalt, zinc and iron(II), have been reported to be present in AKs from some Gram-negative bacteria. Native zinc-containing AK from Desulfovibrio gigas was purified to homogeneity and crystallized. The crystals diffracted to beyond 1.8 A resolution. Furthermore, cobalt- and iron-containing crystal forms of recombinant AK were also obtained and diffracted to 2.0 and 3.0 A resolution, respectively. Zn(2+)-AK and Fe(2+)-AK crystallized in space group I222 with similar unit-cell parameters, whereas Co(2+)-AK crystallized in space group C2; a monomer was present in the asymmetric unit for both the Zn(2+)-AK and Fe(2+)-AK forms and a dimer was present for the Co(2+)-AK form. The structures of the three metal-bound forms of AK will provide new insights into the role and selectivity of the metal in these enzymes.

The localization of APS reductase and bisulfite reductase in Desulfovibrio gigas, D. vulgaris Hildenborough and D. thermophilus was studied by immunoelectron microscopy. Polyclonal antibodies were raised against the purified enzymes from each strain. Cells fixed with formaldehyde/glutaraldehyde were embedded and ultrathin sections were incubated with antibodies and subsequently labeled with protein A-gold. The bisulfite reductase in all three strains and APS reductase in d. gigas and D. vulgaris were found in the cytoplasm. The labeling of d. thermophilus with APS reductase antibodies resulted in a distribution of gold particles over the cytoplasmic membrane region. The localization of the two enzymes is discussed with respect to the mechanism and energetics of dissimilatory sulfate reduction.

This article describes the method and results of our participation in the Critical Assessment of PRediction of Interactions (CAPRI) experiment, using the protein docking program BiGGER (Bimolecular complex Generation with Global Evaluation and Ranking) (Palma et al., Proteins 2000;39:372-384). Of five target complexes (CAPRI targets 2, 4, 5, 6, and 7), only one was successfully predicted (target 6), but BiGGER generated reasonable models for targets 4, 5, and 7, which could have been identified if additional biochemical information had been available.

Three-dimensional protein structures of the xanthine oxidase family show different solutions for the problem of transferring electrons between the flavin adenine dinucleotide (FAD) group and the molybdenum cofactor. In xanthine oxidase all the cofactors he within domains of the same protein chain, whereas in CO dehydrogenase the Fe-S centres, FAD and Mo cofactors are enclosed in separate chains and the enzyme exists as a stable complex of all three. In aldehyde oxidore-ductase, only Fe-S and Mo co-factors are present in a single protein chain. Flavodoxin is docked to aldehyde oxidoreductase to mimic the flavin component on the intramolecular electron transfer chain of aanthine oxidase and CO dehydrogenase and, remarkably, the main features of the electron-transfer pathway are observed.

Hydrogenase from Desulfovibrio desulfuricans (ATCC No. 27774) grown in unenriched and in enriched 61Ni and 57Fe media has been purified to apparent homogeneity. Two fractions of enzymes with hydrogenase activity were separated and were termed hydrogenase I and hydrogenase II. they were shown to have similar molecular weights (77,600 for hydrogenase I and 75,500 for hydrogenase II), to be composed of two polypeptide chains, and to contain Ni and non-heme iron. Because of its higher specific activity (152 versus 97) hydrogenase II was selected for EPR and Mossbauer studies. As isolated, hydrogenase II exhibits an "isotropic" EPR signal at g = 2.02 and a rhombic EPR signal at g = 2.3, 2.2, and 2.0. Isotopic substitution of 61Ni proves that the rhombic signal is due to Ni. Combining the Mossbauer and EPR data, the isotropic g = 2.02 EPR signal was shown to originate from a 3Fe cluster which may have oxygenous or nitrogenous ligands. In addition, the Mossbauer data also revealed two [4Fe-4S]2+ clusters iun each molecule of hydrogenase II. The EPR and Mossbauer data of hydrogenase I were found to be identical to those of hydrogenase II, indicating that both enzymes have common metallic centers.

Adenylyl sulfate (APS) reductase, the key enzyme of the dissimilatory sulfate respiration, catalyzes the reduction of APS (the activated form of sulfate) to sulfite with release of AMP. A spectroscopic study was carried out with the APS reductase purified from the extremely thermophilic sulfate-reducing archaebacterium Archaeoglobus fulgidus DSM 4304. Combined ultraviolet/visible spectroscopy and low temperature electron paramagnetic resonance (EPR) studies were used in order to characterize the active centers and the reactivity towards AMP and sulfite of this enzyme. The A. fulgidus APS reductase is an iron-sulfur flavoprotein containing two distinct [4Fe-4S] clusters (Centers I and II) very similar to the homologous enzyme from Desulfovibrio gigas. Center I, which has a high redox potential, is reduced by AMP and sulfite, and Center II has a very negative redox potential.

In order to utilize sulfate as the terminal electron acceptor, sulfate-reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO4) reductase plays one of the major roles, reducing AdoPSO4 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovibrio gigas. The protein is composed of two non-identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form (approximately 400 kDa). It is an iron-sulfur, flavin-containing protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa. Low-temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered at g = 2.02 and only detectable below 20 K. This signal represented a minor species (0.10-0.25 spins/mol) and showed line broadening in the enzyme isolated from 57Fe-grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal at g values 2.096, 1.940 and 1.890 (reduced Fe-S center I) observable below 30 K and a concomitant decrease in intensity of the g = 2.02 signal were detected. Effects of chemical reductants (ascorbate, H2/hydrogenase-reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modified g values at 2.079, 1.939 and 1.897), and the complete disappearance of the g = 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin-spin-coupled nature (observable below 20 K), indicating the presence of at least two iron-sulfur centers, (centers I and II). Mossbauer studies on 57Fe-enriched D. gigas AdoPSO4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential less than or equal to 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin-type [4Fe-4S] cluster. Center I exhibits spectra with atypical Mossbauer parameters in its reduced state and has a midpoint potential around 0 mV, which is distinct from that of a ferredoxin-type [4Fe-4S] cluster, suggesting a different structure and/or a distinct cluster-ligand environment.