Orange protein from Desulfovibrio alaskensis G20: insights into the Mo-Cu cluster protein-assisted synthesis.

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.

Incorporation of molybdenum in rubredoxin: models for mononuclear molybdenum enzymes.

Molybdenum is found in the active site of enzymes usually coordinated by one or two pyranopterin molecules. Here, we mimic an enzyme with a mononuclear molybdenum-bis pyranopterin center by incorporating molybdenum in rubredoxin. In the molybdenum-substituted rubredoxin, the metal ion is coordinated by four sulfurs from conserved cysteine residues of the apo-rubredoxin and two other exogenous ligands, oxygen and thiol, forming a Mo((VI))-(S-Cys)4(O)(X) complex, where X represents -OH or -SR. The rubredoxin molybdenum center is stabilized in a Mo(VI) oxidation state, but can be reduced to Mo(IV) via Mo(V) by dithionite, being a suitable model for the spectroscopic properties of resting and reduced forms of molybdenum-bis pyranopterin-containing enzymes. Preliminary experiments indicate that the molybdenum site built in rubredoxin can promote oxo transfer reactions, as exemplified with the oxidation of arsenite to arsenate.

Structural redox control in a 7Fe ferredoxin isolated from Desulfovibrio alaskensis.

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.

New hydroxypyridinone-functionalized sepharoses as sorbing agents for hard metal ions.

Two new polymeric matrices functionalized with 3-hydroxy-4-pyridinone chelating units (HP-NH-SEPH and HP-C=NH-SEPH) have been prepared and studied for their chelating ability towards a set of metal ions (e.g. Fe(III), Al(III), and Th(IV)). Both matrices demonstrated excellent ability to complex these metal ions, but HP-NH-SEPH evidenced higher chelating capacity than HP-C=NH-SEPH. The corresponding metal-complex gels presented high stability in the pH range 3-7, and their chelating capacity followed the order, Fe(III)≈Th(IV)>Al(III), in agreement with previously reported thermodynamics of the corresponding monomeric ligand-metal complexes in aqueous solution. These functionalized supports also showed capacity to be regenerated and reused. Thus, there are good perspectives for potential environmental and medical applications of these new metal sorbents.

New tripodal hydroxypyridinone based chelating agents for Fe(III), Al(III) and Ga(III): Synthesis, physico-chemical properties and bioevaluation.

Two new tris-hydroxypyridinone based compounds (KEMPPr(3,4-HP)(3) and KEMPBu(3,4-HP)(3)) have been developed and studied as strong sequestering agents for iron and the group III of metal ions, aimed as potential pharmacological applications on metal-chelation therapy. Their structure is based on the KEMP acid scaffold to which three 3-hydroxy-4-pyridinone chelating moieties are attached via two different size spacers. After the preparation and characterization of the compounds their physico-chemical properties were studied, in relation with their metal binding affinity and lipophilicity. The KEMPPr(3,4-HP)(3) ligand was also bioassayed to evaluate its in vivo metal sequestering capacity from most organs using an animal model overload with (67)Ga. These studies showed that, for both in solution and in vivo conditions, the compounds have higher metal chelating efficacy than Deferriprone, the commercially available iron chelator in medical application, thus some perspectives are envisaged as potential pharmaceutical drug candidates for chelating therapy.