Effects of radical stress on metal complexes of a plant metallothionein: desulfurisation reactions associated with the formation of trans lipids in model membrane.

The effects of radical stress in the biological environment is a very active field of research connecting varies discipline in life science. Thus, a comprehensive vision of all possible reactive species is necessary for contributing to the solution of puzzling questions on free radicals. In this contest, damages to Zn(II) and Cd(II) complexes of a metallothionein from a plant (Quercus suber -QsMT), due to radical stress exposure, were investigated. Metallothioneins (MTs) are low molecular weight, cysteine-rich proteins with high capacity for binding both essential (ZnII and CuI) and xenobiotic (CdII and HgII) metal ions. Metal-MT coordination is mainly achieved by formation of metal-thiolate bonds. Also the contribution of non-proteic ligands, such as chloride and/or sulfide anions, has been recently reported. As almost all the plant MTs, QsMT presents a peculiar amino acid sequence organization consisting of two Cys-rich domains (8 and 6 Cys each), linked by a cysteine-devoid spacer region which includes additional putative ligand (His). Although MTs do not appear to be essential for life, there is mounting evidences for a survival advantage of MT in situations of stress, including exposure to radicals and toxic metals. In support to a MT function in control of oxidative stress, many studies have showed that MT expression increases most dramatically in response to tissue injury, inflammation, tumour and it appears to reduce apoptosis. The reactions of reductive reactive species (H' atoms and eaq-), produced by gamma-irradiation of water, with Zn- and Cd-QsMT were carried out in both aqueous solutions and vesicle suspensions, and were characterized by different approaches. Using a biomimetic model based on unsaturated lipid vesicle suspensions the occurrence of tandem protein/lipid damage was shown The reactions of reductive reactive species with methionine residues and/or sulfur-containing ligands afford diffusible sulfur-centered radicals, which migrate from the aqueous phase to the lipid bilayer and transform the cis double bond of the oleate moiety to the trans isomer. Tailored experiments allowed the reaction mechanism to be defined in some details. The formation of sulfur-centered radicals is accompanied by the modification of the metal-QsMT complexes, which were monitored by various spectroscopic and spectrometric techniques (Raman and ESI-MS). The H' atoms and eaq- attacks on the metal-QsMT aggregates are able to induce significant structural changes such as partial deconstruction and/or rearrangement of the metal clusters, and breaking of the protein backbone. In particular, Cys is among the most sensitive residues towards radical attack, suggesting that the thiolate clusters of both metal-QsMTs act as efficient interceptors of reducing species. The radical-induced effects are dependent on the divalent metal bound.