Stabilization of sulfide cations: mechanisms relevant to oxidation of peptides and proteins containing methionine

Sulfide radical cations (R2S+.) have recently attracted considerable attention. In particular they are implicated in assorted biological electron transfers where they are likely intermediates in biological redox-processes. There is unambigous theoretical and experimental evidence that R2S+. can be stabilized through intramolecular complexation with nucleophiles that are present in neighboring groups. Reactions of this type are of special interest to biology when stabilization of sulfide radical cations derived from methionine, Met(>S+.) occurs in peptides and proteins. The methionine (Met) residues in these biopolymers are susceptible to attack by Reactive Oxygen Species (ROS) during oxidative stress and biological aging. Moreover, the pathogenesis of some neurodegenerative diseases (Alzheimer's, Jacob-Creutzfeld's, and Parkinson's) seems to be strongly linked to the presence in brain tissue of β-amyloid peptide (βAP), human prion protein (hPrP), and an aggregated form of α-synuclein, respectively. These macro- molecules contain methionine(s) with βAP having a Met35 residue in its C-terminal α-helical domain, hPrP having three out of nine Met-residues (namely Met205, Met206, and Met213) located within its α-helical segments, and α-synuclein having four Met-residues. The effective neighboring-group interactions would likely involve nucleophilic functionalities in the side chain of amino acids residues. However, very often heteroatoms in peptide bonds are the only nucleophiles present in the vicinity of Met(>S+.). In this regard, it was recently shown that such interactions play an important role in N-acetylmethione amide and in oligopeptides of the form N-Ac-Gly-Met-Gly and N-Ac-Gly-(Gly)2-Met-(Gly)3. Intramolecularly bonded sulfide radical cations, Met(>S+.), were directly observed in these systems with the bonding partner being either the carbonyl oxygen or the amide nitrogen of a peptide bond. Cyclic dipeptides are suitable model compounds for the study of peptide free radical chemistry. While appearing very small to be models for proteins, they have unique feature of having no terminal groups. This makes them invaluable for studying interactions between side chains and peptide bonds. A small model cyclic dipeptide c-(L-methionyl-L-methionine) 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 emerged from this investigation showed there was an efficient formation of the Met(S N) radicals, in spite of the close proximity of sulfur atoms, located in the side chain of methionine residues, and in spite of the close proximity of sulfur atoms and oxygen atoms, located in the peptide bonds. Moreover, it was observed, for the first time, that formation of Met(S N) radicals involved the hydrogen atom of the peptide bond. In this concerted process, elimination of OH in the form of water involves a simultaneous N-deprotonation from the amide nitrogen, followed by formation of Met(S N) radicals in the form of a thermodynamically favorable five-membered ring. These Met(S N) radicals converted further into intramolecular three-electron bonded Met(S S)+ and Met(S O)+ radical cations via a pH-dependent mechanism. A preference for Met(S+) stabilization in the form of intramolecular three-electron bonded Met(S N) radicals over intermolecular three-electron bonded Met(S S)+ dimeric radical cations was observed in c-(L-Met-D-Met). Lack of intramolecular three-electron bonded Met(S S)+ radical cations illustrates that a close contact between two sulfur atoms is sterically restricted in the D-L enantiomer. Moreover, contrary to c-(L-Met-L-Met), most of Met(S+) radicals derived from c-(L-Met-D-Met) undergo efficient deprotonation in the α-position to the sulfur, yielding carbon-centered α-(alkylthio)alkyl radicals. To support the mechanism, quantum mechanical (DFT) and molecular mechanics (umbrella sampling) calculations were performed that provided thermodynamic parameters for the reactions involved. Our current findings may be useful for the interpretation of data obtained with proteins containing multiple Met residues since they underline the importance of the stabilization of sulfide radical cations by heteroatoms in peptide bonds. 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 for the formation of S N three-electron bonded radical complexes involving the sulfur atom of a methionine residue and the amide groups in adjacent peptide bonds. (authors)