We report on a new method to make nanostructures in aqueous solution at room temperature. We used the protein cytochrome c(3) to catalyze reduction of selenate (SeO42-) to selenium Se-0 by dithionite. Reduction was instantaneous. After a week spherical nanoparticles of red Se-0 (about 50 nm diameter) precipitated, followed by self-assembling into crystalline nanowires, typically 1 mu m long. The nanowires were composed of one strand of spherical particles; thicker strands contained several nanoparticles in parallel.

The contribution of decameric vanadate species to vanadate toxic effects in cardiac muscle was studied following an intravenous administration of a decavanadate solution (1mM total vanadium) in Sparus aurata. Although decameric vanadate is unstable in the assay medium, it decomposes with a half-life time of 16 allowing studying its effects not only in vitro but also in vivo. After 1, 6 and 12h upon decavanadate administration the increase of vanadium in blood plasma, red blood cells and in cardiac mitochondria and cytosol is not affected in comparison to the administration of a metavanadate solution containing labile oxovanadates. Cardiac tissue lipid peroxidation increases up to 20%, 1, 6 and 12h after metavanadate administration, whilst for decavanadate no effects were observed except 1h after treatment (+20%). Metavanadate administration clearly differs from decavanadate by enhancing, 12h after exposure, mitochondrial superoxide dismutase (SOD) activity (+115%) and not affecting catalase (CAT) activity whereas decavanadate increases SOD activity by 20% and decreases (-55%) mitochondrial CAT activity. At early times of exposure, 1 and 6h, the only effect observed upon decavanadate administration was the increase by 20% of SOD activity. In conclusion, decavanadate has a different response pattern of lipid peroxidation and oxidative stress markers, in spite of the same vanadium distribution in cardiac cells observed after decavanadate and metavanadate administration. It is suggested that once formed decameric vanadate species has a different reactivity than vanadate, thus, pointing out that the differential contribution of vanadium oligomers should be taken into account to rationalize in vivo vanadate toxicity.

The blue or Type 1 (T1) copper site of Paracoccus pantotrophus pseudoazurin exhibits significant absorption intensity in both the 450 and 600 nm regions. These are sigma and pi S(Cys) to Cu(2+) charge transfer (CT) transitions. The temperature dependent absorption, EPR, and resonance Raman (rR) vibrations enhanced by these bands indicate that a single species is present at all temperatures. This contrasts the temperature dependent behavior of the T1 center in nitrite reductase [S. Ghosh, X. Xie, A. Dey, Y. Sun, C. Scholes, E. Solomon, Proc. Natl. Acad. Sci. 106 (2009) 4969-4974] which has a thioether ligand that is unconstrained by the protein. The lack of temperature dependence in the T1 site in pseudoazurin indicates the presence of a protein constraint similar to the blue Cu site in plastocyanin where the thioether ligand is constrained at 2.8 angstrom. However, plastocyanin exhibits only pi CT. This spectral difference between pseudoazurin and plastocyanin reflects a coupled distortion of the site where the axial thiorether in pseudoazurin is also constrained, but at a shorter Cu-S(Met) bond length. This leads to an increase in the Cu(2+)-S(Cys) bond length, and the site undergoes a partial tetragonal distortion in pseudoazurin. Thus, its ground state wavefunction has both sigma and pi character in the Cu(2+)-S(Cys) bond. (C) 2009 Elsevier Inc. All rights reserved.

Desulfovibrio gigas desulforedoxin (Dx) consists of two identical peptides, each containing one [Fe-4S] center per monomer. Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli. The three forms of the protein, the two homodimers [Fe(III)/Fe(III)]Dx and [Zn(II)/Zn(II)]Dx, and the heterodimer [Fe(III)/Zn(II)]Dx, can be separated by ion exchange chromatography on the basis of their charge differences. Once separated, the desulforedoxins containing iron can be reduced with added dithionite. For NMR studies, different protein samples were prepared labeled with (15)N or (15)N + (13)C. Spectral assignments were determined for [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx from 3D (15)N TOCSY-HSQC and NOESY-HSQC data, and compared with those reported previously for [Zn(II)/Zn(II)]Dx. Assignments for the (13)C(alpha) shifts were obtained from an HNCA experiment. Comparison of (1)H-(15)N HSQC spectra of [Zn(II)/Zn(II)]Dx, [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx revealed that the pseudocontact shifts in [Fe(II)/Zn(II)]Dx can be decomposed into inter- and intramonomer components, which, when summed, accurately predict the observed pseudocontact shifts observed for [Fe(II)/Fe(II)]Dx. The degree of linearity observed in the pseudocontact shifts for residues >/=8.5 A from the metal center indicates that the replacement of Fe(II) by Zn(II) produces little or no change in the structure of Dx. The results suggest a general strategy for the analysis of NMR spectra of homo-oligomeric proteins in which a paramagnetic center introduced into a single subunit is used to break the magnetic symmetry and make it possible to obtain distance constraints (both pseudocontact and NOE) between subunits.