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A novel metalloprotein containing a unique [SMoSCuSMoS] 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. [ABSTRACT FROM AUTHOR]

Desulfovibrio alaskensis G20, a sulfate-reducing bacterium, contains an arsRBC2C3 operon that encodes two putative arsenate reductases, DaG20_ArsC2 and DaG20_ArsC3. In this study, resistance assays in E. coli transformed with plasmids containing either of the two recombinant arsenate reductases, showed that only DaG20_ArsC3 is functional and able to confer arsenate resistance. Kinetic studies revealed that this enzyme uses thioredoxin as electron donor and therefore belongs to Staphylococcus aureus plasmid pI258 and Bacillus subtilis thioredoxin-coupled arsenate reductases family. Both enzymes from this family contain a potassium-binding site, but only in Sa_ArsC does potassium actually binds resulting in a lower K. Important differences between the S. aureus and B. subtilis enzymes and DaG20_ArsC3 are observed. DaG20_ArsC3 contains only two (Asn10, Ser33) of the four (Asn10, Ser33, Thr63, Asp65) conserved amino acid residues that form the potassium-binding site and the kinetics is not significantly affected by the presence of either potassium or sulfate ions. Isothermal titration calorimetry measurements confirmed nonspecific binding of K and Na, corroborating the non-relevance of these cations for catalysis. Furthermore, the low K and high k values determined for DaG20_ArsC3 revealed that this enzyme is the most catalytically efficient potassium-independent arsenate reductase described so far and, for the first time indicates that potassium binding is not essential to have low K, for Trx-arsenate reductases. [ABSTRACT FROM AUTHOR]

The orange protein (ORP) isolated from the sulfate-reducing bacterium Desulfovibrio gigas (11.8 kDa) contains a mixed-metal sulfide cluster of the type [SMoSCuSMoS] noncovalently bound to the polypeptide chain. The D. gigas ORP was heterologously produced in Escherichia coli in the apo form. Different strategies were used to reconstitute the metal cluster into apo-ORP and obtain insights into the metal cluster synthesis: (1) incorporation of a synthesized inorganic analogue of the native metal cluster and (2) the in situ synthesis of the metal cluster on the addition to apo-ORP of copper chloride and tetrathiomolybdate or tetrathiotungstate. This latter procedure was successful, and the visible spectrum of the Mo-Cu reconstituted ORP is identical to the one reported for the native protein with absorption maxima at 340 and 480 nm. The H-N heteronuclear single quantum coherence spectra of the reconstituted ORP obtained by strategy 2, in contrast to strategy 1, exhibited large changes, which required sequential assignment in order to identify, by chemical shift differences, the residues affected by the incorporation of the cluster, which is stabilized inside the protein by both electrostatic and hydrophobic interactions. [ABSTRACT FROM AUTHOR]

In this work we present a kinetic study of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and members of the three different classes of superoxide reductases (SORs). SORs from the sulfate-reducing bacteria Desulfovibrio vulgaris ( Dv) and D. gigas ( Dg) were chosen as prototypes of classes I and II, respectively, while SOR from the syphilis spyrochete Treponema pallidum ( Tp) was representative of class III. Our results show evidence for different behaviors of SORs toward electron acceptance, with a trend to specificity for the electron donor and acceptor from the same organism. Comparison of the different k app values, 176.9±25.0 min−1 in the case of the Tp/ Tp electron transfer, 31.8±3.6 min−1 for the Dg/ Dg electron transfer, and 6.9±1.3 min−1 for Dv/ Dv, could suggest an adaptation of the superoxide-mediated electron transfer efficiency to various environmental conditions. We also demonstrate that, in Dg, another iron–sulfur protein, a desulforedoxin, is able to transfer electrons to SOR more efficiently than rubredoxin, with a k app value of 108.8±12.0 min−1, and was then assigned as the potential physiological electron donor in this organism. [ABSTRACT FROM AUTHOR]

Desulfovibrio gigas ferredoxin II ( DgFdII) is a small protein with a polypeptide chain composed of 58 amino acids, containing one Fe3S4 cluster per monomer. Upon studying the redox cycle of this protein, we detected a stable intermediate (FdIIint) with four 1H resonances at 24.1, 20.5, 20.8 and 13.7 ppm. The differences between FdIIox and FdIIint were attributed to conformational changes resulting from the breaking/formation of an internal disulfide bridge. The same 1H NMR methodology used to fully assign the three cysteinyl ligands of the [3Fe–4S] core in the oxidized state ( DgFdIIox) was used here for the assignment of the same three ligands in the intermediate state ( DgFdIIint). The spin-coupling model used for the oxidized form of DgFdII where magnetic exchange coupling constants of around 300 cm−1 and hyperfine coupling constants equal to 1 MHz for all the three iron centres were found, does not explain the isotropic shift temperature dependence for the three cysteinyl cluster ligands in DgFdIIint. This study, together with the spin delocalization mechanism proposed here for DgFdIIint, allows the detection of structural modifications at the [3Fe-4S] cluster in DgFdIIox and DgFdIIint. [ABSTRACT FROM AUTHOR]