Analysis of the soybean metallothionein system under free radical stress: protein modification connected to lipid membrane damage

Abstract

Metallothioneins are small Cys-rich peptides capable of coordinating metal ions, and proposed to be involved in radical stress. The four Zn(II)–GmMT complexes of soybean (Glycine max) were recombinantly synthesised and exposed to oxidative (HO˙) and reductive (H˙ atoms and e_aq⁻) stress conditions. Gamma-irradiation was used to simulate the endogenous formation of the reactive species in both aqueous solutions and unsaturated lipid vesicle suspensions, a biomimetic model that showed that tandem protein/lipid damage occurs, in particular under reductive radical stress. This is due to the formation of diffusible sulphur-centred radicals, which migrate from the aqueous phase to the lipid bilayer and are thus able to transform the cis double bond of the oleate moiety into the trans isomer. Among the amino acid residues present in GmMTs, Cys is one of the most sensitive residues towards the attack of free radicals, thus suggesting metal-clusters to be good interceptors of free radicals. Also Met, Tyr and Phe residues are sensitive amino acid sites of attack under both oxidative and reductive conditions. The modification of the Zn(II)–GmMT complexes, in particular isoform 2, was monitored by Raman spectroscopy and mass spectrometry. Free radical stress on the Zn(II)–GmMT complexes is able to induce significant structural changes such as partial deconstruction and/or rearrangement of the metal clusters, but not the complete demetallation of the proteins nor breaking of the backbone, thus confirming their capability to act as protectors under free radical stress conditions.