Superoxide reductases catalyze the monovalent reduction of superoxide anion to hydrogen peroxide. Spectroscopic evidence for the formation of a dinuclear cyano-bridged adduct after K3Fe-(CN)(6) oxidation of the superoxide reductases neelaredoxin from Treponema pallidum and desulfoferrodoxin from Desulfovibrio vulgaris was reported. Oxidation with K3Fe(CN)(6) reveals a band in the near-IR with lambda(max) at 1020 nm, coupled with an increase of the iron content by almost 2-fold. Fourier transform infrared spectroscopy provided additional evidence with CN-stretching vibrations at 2095, 2025-2030, and 2047 cm(-1), assigned to a ferrocyanide adduct of the enzyme. Interestingly, the low-temperature electronic paramagnetic resonance (EPR) spectra of oxidized TpNIr reveal at least three different species indicating structural heterogeneity in the coordination environment of the active site Fe ion. Given the likely 6-coordinate geometry of the active site Fe3+ ion in the ferrocyanide adduct, we propose that the rhombic EPR species can serve as a model of a hexacoordinate form of the active site.

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. Mossbauer 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. Further-more, 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. (C) 2009 Published by Elsevier Inc.

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.