Decameric vanadate species (V10) inhibit the rate and the extent of G-actin polymerization with an IC50 of 68 ± 22 lM and 17 ± 2 lM, respectively, whilst they induce F-actin depolymerization at a lower extent. On contrary, no effect on actin polymerization and depolymerization was detected for 2 mM concentration of ''metavanadate'' solution that contains ortho and metavanadate species, as observed by combining kinetic with 51 V NMR spectroscopy studies. Although at 25°C, decameric vanadate (10 lM) is unstable in the assay medium, and decomposes following a first-order kinetic, in the presence of G-actin (up to 8 lM), the half-life increases 5-fold (from 5 to 27 h). However, the addition of ATP (0.2 mM) in the medium not only prevents the inhibition of G-actin polymerization by V10 but it also decreases the half-life of decomposition of decameric vanadate species from 27 to 10 h. Decameric vanadate is also stabilized by the sarcoplasmic reticulum vesicles, which raise the half-life time from 5 to 18 h whereas no effects were observed in the presence of phosphatidylcholine liposomes, myosin or G-actin alone. It is proposed that the ''decavanadate'' interaction with G-actin, favored by the G-actin polymerization, stabilizes decameric vanadate species and induces inhibition of G-actin polymerization. Decameric vanadate stabilization by cytoskeletal and transmembrane proteins can account, at least in part, for decavanadate toxicity reported in the evaluation of vanadium (V) effects in biological systems.

Incubation of actin with decavanadate induces cysteine oxidation and oxidovanadium(IV) formation. The studies were performed combining kinetic with spectroscopic (NMR and EPR) methodologies. Although decavanadate is converted to labile oxovanadates, the rate of deoligomerization can be very slow (half-life time of 5.4 h, at 25 • C, with a first order kinetics), which effectively allows decavanadate to exist for some time under experimental conditions. It was observed that decavanadate inhibits F-actin-stimulated myosin ATPase activity with an IC 50 of 0.8 mM V 10 species, whereas 50 mM of vanadate or oxidovanadium(IV) only inhibits enzyme activity up to 25%. Moreover, from these three vanadium forms, only decavanadate induces the oxidation of the so called "fast" cysteines (or exposed cysteine, Cys-374) when the enzyme is in the polymerized and active form, F-actin, with an IC 50 of 1 mM V 10 species. Decavanadate exposition to F-and G-actin (monomeric form) promotes vanadate reduction since a typical EPR oxidovanadium(IV) spectrum was observed. Upon observation that V 10 reduces to oxidovanadium(IV), it is proposed that this cation interacts with G-actin (K d of 7.48 ± 1.11 mM), and with F-actin (K d = 43.05 ± 5.34 mM) with 1:1 and 4:1 stoichiometries, respectively, as observed by EPR upon protein titration with oxidovanadium(IV). The interaction of oxidovanadium(IV) with the protein may occur close to the ATP binding site of actin, eventually with lysine-336 and 3 water molecules.

N2OR has been found to have two structural forms of its tetranuclear copper active site, the 4CuS CuZ* form and the 4Cu2S CuZ form. EPR, resonance Raman, and MCD spectroscopies have been used to determine the redox states of these sites under different reductant conditions, showing that the CuZ* site accesses the 1-hole and fully reduced redox states while the CuZ site accesses the 2-hole and 1-hole redox states. Single turnover reactions of N2OR for CuZ and CuZ* poised in these redox states and steady state turnover assays with different proportions of CuZ and CuZ* show that only fully reduced CuZ* is catalytically competent in rapid turnover with N2O.

EXAFS and XANES experiments were used to assess decavanadate interplay with actin, in both the globular and polymerized forms, under different conditions of pH, temperature, ionic strength, and presence of ATP. This approach allowed us to simultaneously probe, for the first time, all vanadium species present in the system. It was established that decavanadate interacts with G-actin, triggering a protein conformational reorientation that induces oxidation of the cysteine core residues and oxidovanadium (V) formation. The local environment of vanadium's absorbing center in the [decavanadate-protein] adducts was determined, a V-S coordination having been verified experimentally. The variations induced in decavanadate's EXAFS profile by the presence of actin were found to be almost totally reversed by the addition of ATP, which constitutes a solid proof of decavanadate interaction with the protein at its ATP binding site. Additionally, a weak decavanadate interplay with F-actin was suggested to take place, through a mechanism different from that inferred for globular actin. These findings have important consequences for the understanding, at a molecular level, of the significant biological activities of decavanadate and similar polyoxometalates, aiming at potential pharmacological applications.

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Although the number of papers about ''vanadium'' has doubled in the last decade, the studies about ''vanadium and actin'' are scarce. In the present review, the effects of vanadyl, vanadate and decavanadate on actin structure and function are compared. Decavanadate 51 V NMR signals, at À516 ppm, broadened and decreased in intensity upon actin titration, whereas no effects were observed for vanadate monomers, at À560 ppm. Decavanadate is the only species inducing actin cysteine oxidation and vanadyl formation, both processes being prevented by the natural ligand of the protein, ATP. Vanadyl titration with monomeric actin (G-actin), analysed by EPR spectroscopy, reveals a 1 : 1 binding stoichiometry and a K d of 7.5 mM À1 . Both decavanadate and vanadyl inhibited G-actin polymerization into actin filaments (F-actin), with a IC 50 of 68 and 300 mM, respectively, as analysed by light scattering assays, whereas no effects were detected for vanadate up to 2 mM. However, only vanadyl (up to 200 mM) induces 100% of G-actin intrinsic fluorescence quenching, whereas decavanadate shows an opposite effect, which suggests the presence of vanadyl high affinity actin binding sites. Decavanadate increases (2.6-fold) the actin hydrophobic surface, evaluated using the ANSA probe, whereas vanadyl decreases it (15%). Both vanadium species increased the e-ATP exchange rate (k = 6.5 Â 10 À3 s À1 and 4.47 Â 10 À3 s À1 for decavanadate and vanadyl, respectively). Finally, 1 H NMR spectra of G-actin treated with 0.1 mM decavanadate clearly indicate that major alterations occur in protein structure, which are much less visible in the presence of ATP, confirming the preventive effect of the nucleotide on the decavanadate interaction with the protein. Putting it all together, it is suggested that actin, which is involved in many cellular processes, might be a potential target not only for decavanadate but above all for vanadyl. By affecting actin structure and function, vanadium can regulate many cellular processes of great physiological significance.

Cytochrome b is the main electron acceptor of cytochrome b reductase. The interacting domain between both human proteins has been unidentified up to date and very little is known about its redox properties modulation upon complex formation. In this article, we characterized the protein/protein interacting interface by solution NMR and molecular docking. In addition, upon complex formation, we measured an increase of cytochrome b reductase flavin autofluorescence that was dependent upon the presence of cytochrome b Data analysis of these results allowed us to calculate a dissociation constant value between proteins of 0.5±0.1μM and a 1:1 stoichiometry for the complex formation. In addition, a 30mV negative shift of cytochrome b reductase redox potential in presence of cytochrome b was also measured. These experiments suggest that the FAD group of cytochrome b reductase increase its solvent exposition upon complex formation promoting an efficient electron transfer between the proteins.