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A novel metalloprotein containing a unique [S2MoS2CuS2MoS2](3-) cluster, designated as Orange Protein (ORP), was isolated for the first time from Desulfovibrio gigas, a sulphate reducer. The orp operon is conserved in almost all sequenced Desulfovibrio genomes and in other anaerobic bacteria, however, so far D. gigas ORP had been the only ORP characterized in the literature. In this work, the purification of another ORP isolated form Desulfovibrio alaskensis G20 is reported. The native protein is monomeric (12443.8 ± 0.1 Da by ESI-MS) and contains also a MoCu cluster with characteristic absorption bands at 337 and 480 nm, assigned to S-Mo charge transfer bands. Desulfovibrio alaskensis G20 recombinant protein was obtained in the apo-form from E. coli. Cluster reconstitution studies and UV-visible titrations with tetrathiomolybdate of the apo-ORP incubated with Cu ions indicate that the cluster is incorporated in a protein metal-assisted synthetic mode and the protein favors the 2Mo:1Cu stoichiometry. In Desulfovibrio alaskensis G20, the orp genes are encoded by a polycistronic unit composed of six genes whereas in Desulfovibrio vulgaris Hildenborough the same genes are organized into two divergent operons, although the composition in genes is similar. The gene expression of ORP (Dde_3198) increased 6.6 ± 0.5 times when molybdate was added to the growth medium but was not affected by Cu(II) addition, suggesting an involvement in molybdenum metabolism directly or indirectly in these anaerobic bacteria.

The redox behaviour of a ferredoxin (Fd) from Desulfovibrio alaskensis was characterized by electrochemistry. The protein was isolated and purified, and showed to be a tetramer containing one [3Fe-4S] and one [4Fe-4S] centre. This ferredoxin has high homology with FdI from Desulfovibrio vulgaris Miyazaki and Hildenborough and FdIII from Desulfovibrio africanus. From differential pulse voltammetry the following signals were identified: [3Fe-4S](+1/0) (E(0')=-158±5mV); [4Fe-4S](+2/+1) (E(0')=-474±5mV) and [3Fe-4S](0/-2) (E(0')=-660±5mV). The effect of pH on these signals showed that the reduced [3Fe-4S](0) cluster has a pK'(red)(')=5.1±0.1, the [4Fe-4S](+2/+1) centre is pH independent, and the [3Fe-4S](0/-2) reduction is accompanied by the binding of two protons. The ability of the [3Fe-4S](0) cluster to be converted into a new [4Fe-4S] cluster was proven. The redox potential of the original [4Fe-4S] centre showed to be dependent on the formation of the new [4Fe-4S] centre, which results in a positive shift (ca. 70mV) of the redox potential of the original centre. Being most [Fe-S] proteins involved in electron transport processes, the electrochemical characterization of their clusters is essential to understand their biological function. Complementary EPR studies were performed., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The isolation and characterization of a new metalloprotein containing Cu and Fe atoms is reported. The as-isolated Cu-Fe protein shows an UV-visible spectrum with absorption bands at 320 nm, 409 nm and 615 nm. Molecular mass of the native protein along with denaturating electrophoresis and mass spectrometry data show that this protein is a multimer consisting of 14+/-1 subunits of 15254.3+/-7.6 Da. Mössbauer spectroscopy data of the as-isolated Cu-Fe protein is consistent with the presence of [2Fe-2S](2+) centers. Data interpretation of the dithionite reduced protein suggest that the metallic cluster could be constituted by two ferromagnetically coupled [2Fe-2S](+) spin delocalized pairs. The biochemical properties of the Cu-Fe protein are similar to the recently reported molybdenum resistance associated protein from Desulfovibrio, D. alaskensis. Furthermore, a BLAST search from the DNA deduced amino acid sequence shows that the Cu-Fe protein has homology with proteins annotated as zinc resistance associated proteins from Desulfovibrio, D. alaskensis, D. vulgaris Hildenborough, D. piger ATCC 29098. These facts suggest a possible role of the Cu-Fe protein in metal tolerance.

The characterization of a novel Mo-Fe protein (MorP) associated with a system that responds to Mo in Desulfovibrio alaskensis is reported. Biochemical characterization shows that MorP is a periplasmic homomultimer of high molecular weight (260 +/- 13 kDa) consisting of 16-18 monomers of 15321.1 +/- 0.5 Da. The UV/visible absorption spectrum of the as-isolated protein shows absorption peaks around 280, 320, and 570 nm with extinction coefficients of 18700, 12800, and 5000 M(-1) cm(-1), respectively. Metal content, EXAFS data and DFT calculations support the presence of a Mo-2S-[2Fe-2S]-2S-Mo cluster never reported before. Analysis of the available genomes from Desulfovibrio species shows that the MorP encoding gene is located downstream of a sensor and a regulator gene. This type of gene arrangement, called two component system, is used by the cell to regulate diverse physiological processes in response to changes in environmental conditions. Increase of both gene expression and protein production was observed when cells were cultured in the presence of 45 microM molybdenum. Involvement of this system in Mo tolerance of sulfate reducing bacteria is proposed.

The cytochrome c nitrite reductase is isolated from the membranes of the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 as a heterooligomeric complex composed by two subunits (61 kDa and 19 kDa) containing c-type hemes, encoded by the genes nrfA and nrfH, respectively. The extracted complex has in average a 2NrfA:1NrfH composition. The separation of ccNiR subunits from one another is accomplished by gel filtration chromatography in the presence of SDS. The amino-acid sequence and biochemical subunits characterization show that NrfA contains five hemes and NrfH four hemes. These considerations enabled the revision of a vast amount of existing spectroscopic data on the NrfHA complex that was not originally well interpreted due to the lack of knowledge on the heme content and the oligomeric enzyme status. Based on EPR and Mössbauer parameters and their correlation to structural information recently obtained from X-ray crystallography on the NrfA structure [Cunha, C.A., Macieira, S., Dias, J.M., Almeida, M.G., Gonçalves, L.M.L., Costa, C., Lampreia, J., Huber, R., Moura, J.J.G., Moura, I. & Romão, M. (2003) J. Biol. Chem. 278, 17455-17465], we propose the full assignment of midpoint reduction potentials values to the individual hemes. NrfA contains the high-spin catalytic site (-80 mV) as well as a quite unusual high reduction potential (+150 mV)/low-spin bis-His coordinated heme, considered to be the site where electrons enter. In addition, the reassessment of the spectroscopic data allowed the first partial spectroscopic characterization of the NrfH subunit. The four NrfH hemes are all in a low-spin state (S = 1/2). One of them has a gmax at 3.55, characteristic of bis-histidinyl iron ligands in a noncoplanar arrangement, and has a positive reduction potential.

Desulfovibrio gigas desulforedoxin (Dx) consists of two identical peptides, each containing one [Fe-4S] center per monomer. Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli. The three forms of the protein, the two homodimers [Fe(III)/Fe(III)]Dx and [Zn(II)/Zn(II)]Dx, and the heterodimer [Fe(III)/Zn(II)]Dx, can be separated by ion exchange chromatography on the basis of their charge differences. Once separated, the desulforedoxins containing iron can be reduced with added dithionite. For NMR studies, different protein samples were prepared labeled with (15)N or (15)N + (13)C. Spectral assignments were determined for [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx from 3D (15)N TOCSY-HSQC and NOESY-HSQC data, and compared with those reported previously for [Zn(II)/Zn(II)]Dx. Assignments for the (13)C(alpha) shifts were obtained from an HNCA experiment. Comparison of (1)H-(15)N HSQC spectra of [Zn(II)/Zn(II)]Dx, [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx revealed that the pseudocontact shifts in [Fe(II)/Zn(II)]Dx can be decomposed into inter- and intramonomer components, which, when summed, accurately predict the observed pseudocontact shifts observed for [Fe(II)/Fe(II)]Dx. The degree of linearity observed in the pseudocontact shifts for residues >/=8.5 A from the metal center indicates that the replacement of Fe(II) by Zn(II) produces little or no change in the structure of Dx. The results suggest a general strategy for the analysis of NMR spectra of homo-oligomeric proteins in which a paramagnetic center introduced into a single subunit is used to break the magnetic symmetry and make it possible to obtain distance constraints (both pseudocontact and NOE) between subunits.

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 (≈ 400 kDa). It is an iron-sulfur, ravincontaining 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). Mössbauer studies on 57Fe-enriched D. gigas AdoPSO4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential ≤ 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin-type [4Fe-4S] cluster. Center I exhibits spectra with atypical Mössbauer 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. [ABSTRACT FROM AUTHOR]

The kinetics of electron transfer between the Desulfovibro gigas hydrogenase and several electron-transfer proteins from Desulfovibrio species were investigated by cyclic voltammetry, square-wave voltammetry and chronoamperometry. The cytochrome C3 from Desulfovibrio vulgaris (Hildenborough). Desulfovibrio desulfuricans (Norway 4), Desulfovibrio desulfuricans (American Type Culture Collection 27774) and D. gigas (NCIB 9332) were used as redox carriers. They differ in their redox potentials and isoelectric point. Depending on the pH, all the reduced forms of these cytochromes were effective in electron exchange with hydrogenase. Other small electron-transfer proteins such as ferredoxin I, ferredoxin II and rubredoxin from D. gigas were tentatively used redox carriers. Only ferredoxin II was effective in mediating electron exchange between hydrogenase and the working electrode. The second-order rate constants k for the reaction between reduced proteins and hydrogenase were calculated based on the theory of the simplest electrocatalytic mechanism [Moreno, C., Costa, C., Moura, I., Le Gall, J., Liu, M. Y., Payne, W.J., van Dijk, C. & Moura. J.J.G. (1193) Eur. J.. Biochem. 212. 79-86] and the results obtained by cyclic voltammetry were compared with those obtained by chronoamperometry. Values for k of 105-106 M&sup-1; s&sup-1; (cytochrome c1 as electron carrier) and 104M&sup-1; s&sup-1; (ferredoxin II as the electron carrier) were determined. The rate-constant values are discussed in terms of the existence of an electrostatic interaction between the electrode surface and the redox carrier and between the redox carrier and a positively charged part of the enzyme. [ABSTRACT FROM AUTHOR]