A gene encoding Bfr (bacterioferritin) was identified and isolated from the genome of Desulfovibrio vulgaris cells, and overexpressed in Escherichia coli. In vitro, H(2)O(2) oxidizes Fe(2+) ions at much higher reaction rates than O(2). The H(2)O(2) oxidation of two Fe(2+) ions was proven by Mössbauer spectroscopy of rapid freeze-quenched samples. On the basis of the Mössbauer parameters of the intermediate species we propose that D. vulgaris Bfr follows a mineralization mechanism similar to the one reported for vertebrate H-type ferritins subunits, in which a diferrous centre at the ferroxidase site is oxidized to diferric intermediate species, that are subsequently translocated into the inner nanocavity. D. vulgaris recombinant Bfr oxidizes and stores up to 600 iron atoms per protein. This Bfr is able to bind DNA and protect it against hydroxyl radical and DNase deleterious effects. The use of H(2)O(2) as an oxidant, combined with the DNA binding and protection activities, seems to indicate a DPS (DNA-binding protein from starved cells)-like role for D. vulgaris Bfr.

Dps proteins (DNA-binding protein from starved cells) are hollow-sphere-shaped, dodecameric enzymes found in bacteria and archaeal species.They can oxidize ferrous iron in acontrolled manner using hydrogen peroxide or molecular oxygen as co-substrate,a nd most of them confer physical protection through DNAb inding.O xidizedi ron is stored, as am ineral core,i nacentral cavity.D irect evidence is now provided that, furthermore,D ps proteins containing small mineral cores can oxidize and mineralize toxic ferrous ions in anaerobic conditions and in the absence of any additional aqueous oxidant co-substrate.Dps proteins containing cores of 24 irons per dodecamer can oxidize about 5f errous irons per dodecamer,w ith that number approximately doubling for protein particles containing in average 96 irons per protein.This additional activity carries importance as it can be ad etoxification mechanism present during anaerobic or oxygen-limited growth conditions.

DNA-binding proteins from starved cells (Dps) are members of the ferritin family of proteins found in prokaryotes, with hollow rounded cube-like structures, composed of twelve equal subunits. These protein nanocages are bifunctional enzymes that protect the cell from the harmful reaction of iron and peroxide (Fenton reaction), thus preventing DNA damage by oxidative stress. Ferrous ions are oxidized at specific iron binding sites in the presence of the oxidant and stored in its cavity that can accommodate up to ca. 500 iron atoms. DNA binding properties of Dps are associated with the N-terminal, positive charge rich, extensions that can promote DNA binding and condensation, apparently by a cooperative binding mechanism. Here, we describe the binding and protection activities of Marinobacter hydrocarbonoclasticus Dps using Electrophoretic Mobility Shift Essays (EMSA), and Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. While no DNA condensation was observed in the tested conditions, it was possible to determine a Dps-DNA complex formation with an apparent dissociation constant of 5.9 ± 1.0 µM and a Hill coefficient of 1.2 ± 0.1. This interaction is suppressed by the inclusion of a single negative charge in the N-terminal region by point mutation. In Dps proteins containing a ferric mineral core (above 96 Fe/protein) DNA binding was impaired. SRCD data clearly showed that no significant modification existed either in secondary structure or protein stability of WT, Q14E variant and core containing proteins. It was, however, interesting to note that, in our experimental conditions, thermal denaturation induced protein aggregation that caused artifacts in thermal denaturation curves, which were dependent on radiation flux and vertical arrangement of the CD cell.

Organisms are often exposed to different types of ionizing radiation that, directly or not, will promote damage to DNA molecules and/or other cellular structures. Because of that, organisms developed a wide range of response mechanisms to deal with these threats. Endonuclease III is one of the enzymes responsible to detect and repair oxidized pyrimidine base lesions. However, the effect of radiation on the structure/function of these enzymes is not clear yet. Here, we demonstrate the effect of UV-C radiation on E. coli endonuclease III through several techniques, namely UV-visible, fluorescence and Mössbauer spectroscopies, as well as SDS-PAGE and electrophoretic mobility shift assay. We demonstrate that irradiation with a UV-C source has dramatic consequences on the absorption, fluorescence, structure and functionality of the protein, affecting its [4Fe-4S] cluster and its DNA-binding ability, which results in its inactivation. An UV-C radiation-induced conversion of the [4Fe-4S](2+) into a [2Fe-2S](2+) was observed for the first time and proven by Mössbauer and UV-visible analysis. This work also shows that the DNA-binding capability of endonuclease III is highly dependent of the nuclearity of the endogenous iron-sulfur cluster. Thus, from our point of view, in a cellular context, these results strengthen the argument that cellular sensitivity to radiation can also be due to loss of radiation-induced damage repair ability.