Reversible Intramolecular Hydrogen Transfer between Protein Cysteine Thiyl Radicals and ^αC−H Bonds in Insulin: Control of Selectivity by Secondary Structure

Abstract

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.

Mobile protons versus mobile radicals: Gas-phase unimolecular chemistry of radical cations of cysteine-containing peptides

“…The side-chain thiolate radicals of cysteine residues represent an important class of radicals in peptide and protein chemistry that are involved in a number of enzyme reactions [24] and are implicated in protein damage [25]. Recent work by Schoeneich's group has demonstrated that the side-chain thiolate radicals of cysteine residues can abstract hydrogen atoms from amino acids intermolecularly and also intramolecularly from adjacent amino acid residues [67][68][69][70][71][72]. Given the potential importance of these reactions in protein damage, as well as these being excellent model systems to examine side-chain-to-backbone HAT in gas-phase peptides, here we use a combination of multistage mass spectrometry experiments, collision-induced dissociation, deuterium labeling, and density functional theory (DFT) calculations to study the fragmentation reactions of regiospecifically generated radical cations of simple cysteine-containing di-and tripeptides.…”

Photolysis of Recombinant Human Insulin in the Solid State: Formation of a Dithiohemiacetal Product at the C-Terminal Disulfide Bond

“…The deuterium content of peptide ions and their fragments was determined from the differences between the average mass of a covalently deuterated peptide and the average mass of the corresponding fully protonated peptide, according to our published protocol (22). The average masses were calculated from centroided isotopic distributions.…”

“…The distribution of deuterium incorporation was obtained after isotopic correction by subtracting the isotope abundance distribution in the product formed during UV-irradiation in H 2 O from the isotope abundance distribution of the same product generated in D 2 O. This variation of the isotopic distribution between the experiments performed in deuterium oxide solution and water is given by the variation of the percent base peak intensity (%ΔBPI) (22).…”

“…4). In solution, product III is formed along with an isobaric product IV, corresponding to a crosslink between Cys[B19] and Tyr[A19] (22). Most of the b and y fragment ions characteristic of III and IV are identical.…”

“…The disulfide bond is sensitive to light-induced degradation, where recent studies with model peptide have indicated the formation of a series of novel photoproducts such as dithiohemiacetals, and thioethers (18)(19)(20)(21). Additionally, in a previous publication, we investigated in particular the mechanisms of photodegradation of human insulin in solution through the formation of S-centered radicals, where evidence for chemical cross-linking between the A-and B-chain was obtained, together with significant extent of covalent H/D exchange when experiments were carried out in D 2 O (22). Evidence for dithiohemiacetal formation was presented also for the photoirradiation of an immunoglobulin, IgG1, in solution (18).…”