Stabilization of Sulfide Radical Cations through Complexation with the Peptide Bond:  Mechanisms Relevant to Oxidation of Proteins Containing Multiple Methionine Residues

Abstract

The recent study on the *OH-induced oxidation of calmodulin, a regulatory "calcium sensor" protein containing nine methionine (Met) residues, has supported the first experimental evidence in a protein for the formation of S therefore N three-electron bonded radical complexes involving the sulfur atom of a methionine residue and the amide groups in adjacent peptide bonds. To characterize reactions of oxidized methionine residues in proteins containing multiple methionine residues in more detail, in the current study, a small model cyclic dipeptide, c-(L-Met-L-Met), was oxidized by *OH radicals generated via pulse radiolysis and the ensuing reactive intermediates were monitored by time-resolved UV-vis spectroscopic and conductometric techniques. The picture that emerges from this investigation shows there is an efficient formation of the Met (S therefore N) radicals, in spite of the close proximity of two sulfur atoms, located in the side chains of methionine residues, and in spite of the close proximity of sulfur atoms and oxygen atoms, located in the peptide bonds. Moreover, it is shown, for the first time, that the formation of Met(S therefore N) radicals can proceed directly, via H+-transfer, with the involvement of hydrogen from the peptide bond to an intermediary hydroxysulfuranyl radical. Ultimately, the Met(S therefore N) radicals decayed via two different pH-dependent reaction pathways, (i) conversion into sulfur-sulfur, intramolecular, three-electron-bonded radical cations and (ii) a proposed hydrolytic cleavage of the protonated form of the intramolecular, three-electron-bonded radicals [Met(S therefore N)/Met(S therefore NH)+] followed by electron transfer and decarboxylation. Surprisingly, also alpha-(alkylthio)alkyl radicals enter the latter mechanism in a pH-dependent manner. Density functional theory computations were performed on the model c-(L-Met-Gly) and its radicals in order to obtain optimizations and energies to aid in the interpretation of the experiments on c-(L-Met-L-Met).

Gene Expression and Physiological Role of Pseudomonas aeruginosa Methionine Sulfoxide Reductases during Oxidative Stress

“…Superoxide anion-mediated oxidation of Met residues in proteins and free Met has been reported (53,54). In other bacteria, inactivation of msr genes has been shown to increase sensitivity to the superoxide generator paraquat (53); therefore, the physiological roles of P. aeruginosa msrA and msrB in superoxide stress protection were evaluated.…”

Conformational Influence on the Type of Stabilization of Sulfur Radical Cations in Cyclic Peptides

“…[11] A study was recently undertaken in which the cyclic peptide cyclo-l-Met-l-Met was used as a model for proteins with multiple methionine residues. [12] Even though c-(l-Met-l-Met) appears too small to be a protein model, this cyclic dipeptide has the attractive feature that it has no terminal groups. Hence, stabilization of sulfur radical cations can come solely from interactions with lone pairs of electrons in side chains, or with those on heteroatoms associated with the peptide bonds.…”

“…In this small model peptide, sulfur radical cations were stabilized both by intramolecular sulfur-nitrogen bonding with amide nitrogen atoms in the peptide bonds and by intramolecular bonding with the unoxidized sulfur atom in the side chain of the other methionine. [12] In the current study, two other cyclic dipeptides were synthesized so that we could look more closely at the conditions under which sulfur radical cations in methionines can be stabilized by lone pairs of electrons from neighboring sulfur atoms. The dipeptide cyclo-(d-Met-l-Met) was studied with combined optical and conductometric detection following radiolytic oxidation.…”

A combined quantum mechanical and experimental approach towards chiral diketopiperazine hydroperoxides

“…Although several theoretical studies on diketopiperazines exist in the literature, most of them deal with calculation of spectroscopic properties, such as fragmentation patterns in ESI MS spectra, [24] vibrational and electronic spectra, [25] circular dichroic spectra, [26] confor-F o r P e e r R e v i e w mational analysis [27] and crystal packing. [28] Only a couple of references treated reaction pathways such as radical formation of methionine-derived diketopiperazines as calmodulin mimics, [29] formation of imidazo[1,2-a]pyrazine-3,6-diones from diketopiperazines [30] and Pictet-Spengler cyclization of N-2-bromoacetyltetrahydroisoquinoline methyl ester in the presence of primary or secondary amines to the corresponding diketopiperazines. [31] Rauk used DFT methods to calculate kinetics and bond dissociation enthalpies of H-abstraction from symmetrical diketopiperazines by methyl or thiyl radicals.…”