Reversible Intramolecular Hydrogen Transfer between Protein Cysteine Thiyl Radicals and ~(alpha)C-H Bonds in Insulin:Control of Selectivity by Secondary Structure

摘要

The selective oxidative modification of proteins can have significant consequences for structure and function.Here,we show that protein cysteine thiyl radicals(CysS~·)can reversibly abstract hydrogen atoms from the ~(alpha)C-H bonds of selected amino acids in a protein(insulin).CysS~· were generated photolytically through homolysis of cystine and through photoionization of an aromatic residue,followed by one-electron reduction of cystine.Intramolecular hydrogen transfer was monitored through the covalent incorporation of deuterium into specific amino residues.Of 51 insulin amino residues,only six incorporated significant levels of deuterium:Leu(B6),Gly(B8),Ser(B9),Val(B18),Gly(B20),and Cys(A20).All these amino acids are located at the beginning/end or outside of alpha-helices and beta-sheets,in accordance with theory,which predicts that specifically the ~(alpha)C-H bonds of amino acids in these secondary structures have higher homolytic C-H bond dissociation energies compared to the ~(alpha)C-H bonds of amino acids in extended conformations.Through such hydrogen transfer mechanisms,thiyl radicals are able to catalyze the oxidation of amino acids in a protein through oxidants,which would not necessary directly react with these amino acids.This feature has important consequences for protein stability under conditions of oxidative stress and/or protein production in pharmaceutical biotechnology.