Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anion O2- into hydrogen peroxide and water. Treponema pallidum (Tp), the syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression in Escherichia coli and purification of the Tp rubredoxin. Spectroscopic characterization of this 6 kDa protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric iron, epsilon=6.9+/-0.4 mM(-1) cm(-1). Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was -76+/-5 mV. Reduced rubredoxin can be efficiently reoxidized upon addition of Na(2)IrCl(6)-oxidized neelaredoxin, in agreement with a direct electron transfer between the two proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized rubredoxin per one molecule of neelaredoxin. In addition, in presence of a steady-state concentration of superoxide anion, the physiological substrate of neelaredoxin, reoxidation of rubredoxin was also observed in presence of catalytic amounts of superoxide reductase, and the rate of rubredoxin reoxidation was shown to be proportional to the concentration of neelaredoxin, in agreement with a bimolecular reaction, with a calculated k(app)=180 min(-1). Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris resulted in a much lower value of k(app)=4.5 min(-1). Altogether, these results demonstrated the existence for a superoxide-mediated electron transfer between rubredoxin and neelaredoxin and confirmed the physiological character of this electron transfer reaction.

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 spirochete 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 kapp 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 kapp value of 108.8+/-12.0 min(-1), and was then assigned as the potential physiological electron donor in this organism.

Superoxide reductases catalyze the monovalent reduction of superoxide anion to hydrogen peroxide. Spectroscopic evidence for the formation of a dinuclear cyano-bridged adduct after K(3)Fe(CN)(6) oxidation of the superoxide reductases neelaredoxin from Treponema pallidum and desulfoferrodoxin from Desulfovibrio vulgaris was reported. Oxidation with K(3)Fe(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 TpNlr 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 Fe(3+) 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 general affinity of the sarcoplasmic reticulum (SR) Ca (2+)-ATPase was examined for three different classes of vanadium coordination complexes including a vanadium(V) compound, pyridine-2,6-dicarboxylatodioxovanadium(V) (PDC-V(V)), and two vanadium(IV) compounds, bis(maltolato)oxovanadium(IV) (BMOV), and an analogue of amavadine, bis( N-hydroxylamidoiminodiacetato)vanadium(IV) (HAIDA-V(IV)). The ability of vanadate to act either as a phosphate analogue or as a transition-state analogue with enzymes' catalysis phosphoryl group transfer suggests that vanadium coordination compounds may reveal mechanistic preferences in these classes of enzymes. Two of these compounds investigated, PDC-V(V) and BMOV, were hydrolytically and oxidatively reactive at neutral pH, and one, HAIDA-V(IV), does not hydrolyze, oxidize, or otherwise decompose to a measurable extent during the enzyme assay. The SR Ca (2+)-ATPase was inhibited by all three of these complexes. The relative order of inhibition was PDC-V(V) > BMOV > vanadate > HAIDA-V(IV), and the IC 50 values were 25, 40, 80, and 325 microM, respectively. Because the observed inhibition is more potent for PDC-V(V) and BMOV than that of oxovanadates, the inhibition cannot be explained by oxovanadate formation during enzyme assays. Furthermore, the hydrolytically and redox stable amavadine analogue HAIDA-V(IV) inhibited the Ca (2+)-ATPase less than oxovanadates. To gauge the importance of the lipid environment, studies of oxidized BMOV in microemulsions were performed and showed that this system remained in the aqueous pool even though PDC-V(V) is able to penetrate lipid interfaces. These findings suggest that the hydrolytic properties of these complexes may be important in the inhibition of the calcium pump. Our results show that two simple coordination complexes with known insulin enhancing effects can invoke a response in calcium homeostasis and the regulation of muscle contraction through the SR Ca (2+)-ATPase.

Among the biotargets interacting with vanadium is the calcium pump from the sarcoplasmic reticulum (SR). To this end, initial research efforts were launched with two vanadium(V)-citrate complexes, namely (NH(4))(6)[V(2)O(4)(C(6)H(4)O(7))(2)].6H(2)O and (NH(4))(6)[V(2)O(2)(O(2))(2)(C(6)H(4)O(7))(2)].4H(2)O, potentially capable of interacting with the SR calcium pump by combining kinetic studies with (51)V NMR spectroscopy. Upon dissolution in the reaction medium (concentration range: 4-0.5mM), both vanadium(V):citrate (VC) and peroxovanadium(V):citrate (PVC) complexes are partially converted into vanadate oligomers. A 1mM solution of the PVC complex, containing 184microM of the PVC complex, 94microM oxoperoxovanadium(V) (PV) species, 222microM monomeric (V1), 43microM dimeric (V2) and 53microM tetrameric (V4) species, inhibits Ca(2+) accumulation by 75 %, whereas a solution of the VC complex of the same vanadium concentration, containing 98microM of the VC complex, 263microM monomeric (V1), 64microM dimeric (V2) and 92microM tetrameric (V4) species inhibits the calcium pump activity by 33 %. In contrast, a 1 mM metavanadate solution, containing 460microM monomeric (V1), 90.2microM dimeric (V2) and 80microM tetrameric (V4) species, has no effect on Ca(2+) accumulation. The NMR signals from the VC complex (-548.0ppm), PVC complex (-551.5ppm) and PV (-611.1ppm) are broadened upon SR vesicle addition (2.5mg/ml total protein). The relative order for the half width line broadening of the NMR signals, which reflect the interaction with the protein, was found to be V4>PVC>VC>PV>V2=V1=1, with no effect observed for the V1 and V2 signals. Putting it all together the effects of two vanadium(V)-citrate complexes on the modulation of calcium accumulation and ATP hydrolysis by the SR calcium pump reflected the observed variable reactivity into the nature of key species forming upon dissolution of the title complexes in the reaction media.