Sensitized photo-oxidation of plant cytokinin-specific binding protein-Does the environment of the thioether group influence the oxidation reaction? From primary intermediates to stable products

The reactions of protein oxidation play a significant role in many biological processes, especially in diseases development. Therefore, it is important to understand, how the protein molecule behaves in the presence of oxidants. In the present work, photo-oxidation of phytohormone-binding plant protein (VrPhBP) was investigated using light and 3-carboxybenzophenone (3CB) as a sensitizer (one electron oxidant). The protein interacts with the sensitizer in the ground state forming a weak binding complex leading to the presence of bound and free 3CB in solution. The early events and transient species (such as radicals and radical ions) formed during irradiation were characterised by transient spectroscopy showing the formation of the sulphur radical cation Met>S center dot+ (stabilized by (SN)(+))and the tyrosyl radical TyrO(center dot) on VrPhBP. Thus the 3CB excited triplet state was quenched by the Met and Tyr residues and mostly by Met (based on the deconvoluted transient absorption spectra).The presence of a Tyr side chain in the vicinity of a Met residue results in intramolecular electron transfer from Tyr to the Met>S center dot+ radical cation, leading to regeneration of the thioether side chain and formation of TyrO(center dot). The presence of other side chains close to Met, such as Arg or Lys can induce the stabilization of Met>S center dot+ via the formation of two-centered three-electron bonded species (S therefore N)(+). The transient species were additionally confirmed by stable product analysis. Based on SDS-PAGE, chromatography and mass spectrometry, the formation of methionine sulphoxide and Met-3CB adduct was identified together with di-Tyr cross links. On the basis of the experimental results the overall mechanism of VrPhBP photo oxidation, from its early events to the formation of stable products, is described. In addition, a good correlation between the mechanisms of photooxidation of model compounds such as Met derivatives and peptides and those for real biological systems is emphasized.

Automated summary

The study investigates the photo-oxidation process of VrPhBP using transient spectroscopy, revealing the quenching of the 3CB excited triplet state by Met and Tyr residues to form sulphur radical cation and tyrosyl radical. The presence of other side chains like Arg or Lys close to Met was found to stabilize the Met>S center dot+ through the formation of two-centered three-electron bonded species.