Krzysztof Bobrowski

˙OH radical induced decarboxylation of methionine-containing peptides. Influence of peptide sequence and net charge

The ˙OH radical induced oxidation of methionine-containing peptides results in significantly different decarboxylation yields upon variation of the location of the methionine unit with respect to the terminal functions (Met–Gly, Met–Glu, Met–Gly–Gly, Gly–Met–Gly, Gly–Met and Gly–Gly–Met), or with the nature of neighbouring amino acids located at the N-terminus of methionine (Ala–Met, β-Ala-Met, Val-Met, Leu–Met, Ser–Met, Thr–Met, His–Met, γ-Glu–Met, Pro–Met, Gly–Gly–Phe–Met and Tyr–Gly–Gly–Phe–Met). The CO2 yields measured in γ-radiolysis vary from 0%(Met–Gly, Met–Glu, Met–Gly–Gly, Gly–Met–Gly, and Pro–Met) to about 80%(γ–Glu–Met) of the ˙OH radicals available. Mechanistically, the decarboxylation is considered to proceed via an intramolecular ‘outer sphere’ electron transfer from the methionine carboxylate function to the oxidized sulphur function S˙+. An additional N-terminal decarboxylation route exists in γ-Glu–Met which requires assistance by the α-positioned free amino group. Both processes compete with deprotonation ofS˙+ at the carbon atom α-positioned to sulphur. The relative rates of all these competing pathways, and consequently the decarboxylation yields, are shown to depend on (i) the electron inductive properties of substituent groups at the α-carbon of the N-terminal amino acid, (ii) the net electric charge of the peptide molecule, and (iii) the distance between the centres of positive charge (–NH3+ and S˙+).

Sensitized photo-oxidation of sulfur-containing amino acids and peptides in aqueous solution

Abstract Qualitative and quantitative studies of the photo-oxidation of sulfur-containing amino acids and methionine-containing dipeptides and tripeptides in aqueous solution sensitized by 4-carboxybenzophenone (CB) are reviewed. The mechanism of the photo-oxidation reaction was investigated using the techniques of flash photolysis, steady state photolysis and pulse radiolysis. The rate constants for quenching of the CB triplet by twelve sulfur-containing amino acids and six methionine-containing peptides were determined to be in the range 10 8 –10 9 M −1 s −1 for both neutral and alkaline solutions. The amino acids varied in structure, having different numbers of COCH and NH 2 terminal groups and their sulfur atom at different locations relative to the terminal groups. The methionine-containing peptides were MetGly, GlyMet, MetMet MetGlyGly, GlyMetGly and GlyGlyMet. Time-resolved transient spectra accompanying the quenching events were assigned to the triplet states of CB, ketyl radicals of CB, radical anions of CB and radical anions of CB and radical cations derived from the amino acids and peptides. The radical cations identified were intermolecularly (S.·.S) + -bonded cations, intramolecularly (S.·.N) + -bonded cations and an intramolecularly (S.·.S) + -bonded radical cation that was observed in experiments with MetMet. The quantum yields of the transients and their kinetics of formation and decay were measured by flash photolysis. The quantum yields of CO 2 formation were determined by steady state photolysis. Electron transfer from the sulfur atom to the triplet state of the ketone was found to be a primary photochemical step. A detailed mechanism of the CB-sensitized photo-oxidation of sulfur-containing amino acids and methionine-containing peptides, including primary and secondary photoreactions, is proposed and discussed. Within the mechanism, contrasting behavior between the peptides and amino acids as quenchers is emphasized.

Exploring oxidative modifications of tyrosine: An update on mechanisms of formation, advances in analysis and biological consequences

Abstract Protein oxidation is increasingly recognised as an important modulator of biochemical pathways controlling both physiological and pathological processes. While much attention has focused on cysteine modifications in reversible redox signalling, there is increasing evidence that other protein residues are oxidised in vivo with impact on cellular homeostasis and redox signalling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor the formation of oxidised derivatives, which depends on a variety of analytical techniques. While antibody-dependent techniques such as ELISAs are commonly used, these have limitations, and more specific assays based on spectroscopic or spectrometric techniques are required to provide information on the exact residues modified and the nature of the modification. These approaches have helped understanding of the consequences of tyrosine oxidation in biological systems, especially its effects on cell signalling and cell dysfunction, linking to roles in disease. There is mounting evidence that tyrosine oxidation processes are important in vivo and can contribute to cellular pathology.

A reinvestigation of the mechanism of photoreduction of benzophenones by alkyl sulfides

Abstract The photoreduction of benzophenone and 4-carboxybenzophenone by dimethyl sulfide was examined in aqueous, mixed water—acetonitrile (1:1v/v) and acetonitrile solutions by the use of nanosecond laser photolysis. Bimolecular quenching rate constants were determined and were found to be in the range (1.5–4.6)×109 M−1s−1. Electron transfer from the sulfur atom to the triplet state of the benzophenones was found to be a primary photochemical step. This was established by the large values of quenching rate constants and by the observation of free radical ions, i.e. ketyl radical anions and (S∴S)+ radical cations of dimethyl sulfide in aqueous and mixed water-acetonitrile solutions. The overall quantum yields of photoproducts (ketyl radicals and ketyl radical anions) are low (Φtotalketyl=0.26 in aqueous solutions, are in the range 0.16–0.20 in mixed water-acetonitrile solutions, and decrease to less than or equal to 0.01 in pure acetonitrile), suggesting that back electron transfer within the charge-transfer complex to regenerate the reactants is the dominant process.

Photooxidation of Methionine Derivatives by the 4-Carboxybenzophenone Triplet State in Aqueous Solution. Intracomplex Proton Transfer Involving the Amino Group

Abstract— Oxidation of the triplet state of 4‐carboxybenzophenone (CB) by a series of five substituted methionines and three methionine‐containing dipeptides was monitored under laser flash photolysis conditions in aqueous solution. Spectral resolution techniques were employed to follow the concentration profiles of the intermediates formed from the quenching events. From these concentration profiles, quantum yields for the intermediates were determined. Branching ratios were evaluated for the decay of the charge‐transfer complex by the competing processes of back electron transfer, proton transfer and escape of radical ions. The relative prominence of these processes was discussed in terms of the proton‐transfer tendencies of the nominal sulfur‐radical‐cationic species. A systematic decrease was observed in the quantum yields for the escape of radical ions along with a correlated increase in the proton‐transfer yields. The enhanced propensity of the sulfur radical cations to depro‐tonate is due to deprotonation at the carbons adjacent to the sulfur‐cationic site and at the unsubstituted amino groups when present. This scheme was supported by an observed decrease in the yields of dimeric sulfur radical cations with an increase in the electron‐withdrawing abilities of the substituents, making the radical‐cationic species stronger acids. The involvement of protons on the amino groups was implicated by the correlation of the quantum yields of ketyl radical formation in the photochemistry experiments with the rate constants for the reaction of the CB radical anion with the sulfur‐containing substrates in pulse radiolysis experiments.

Kinetic studies on the formation of sulfonyl radicals and their addition to carbon-carbon multiple bonds.

The reactions of α-hydroxyl and α-alkoxyl alkyl radicals with methanesulfonyl chloride (MeSO(2)Cl) have been studied by pulse radiolysis at room temperature. The alkyl radicals were produced by ionizing radiation of N(2)O-saturated aqueous solution containing methanol, ethanol, isopropanol, or tetrahydrofuran. The transient optical absorption spectrum consisted of a broad band in the region 280-380 nm with a maximum at 320 nm typical of the MeSO(2)(•) radical. The rate constants in the interval of 1.7 × 10(7)-2.2 × 10(8) M(-1) s(-1) were assigned to an electron-transfer process that leads to MeSO(2)Cl(•-), subsequently decaying into MeSO(2)(•) radical and Cl(-). The rate constants for the addition of CH(3)SO(2)(•) to acrolein and propiolic acid were found to be 4.9 × 10(9) M(-1) s(-1) and 5.9 × 10(7) M(-1) s(-1), respectively, in aqueous solutions and reversible. The reactivity of tosyl radical (p-CH(3)C(6)H(4)SO(2)(•)) toward a series of alkenes bearing various functional groups was also determined by competition kinetics in benzene. The rate constants for the addition of tosyl radical to alkenes vary in a much narrower range than the rate constants for the reverse reaction. The stabilization of the adduct radical substantially contributes to the increase of the rate constant for the addition of tosyl radical to alkenes and, conversely, retards the β-elimination of tosyl radical.

The use of the methods of radiolysis to explore the mechanisms of free radical modifications in proteins.

The method of radiolysis is based upon the interaction of ionising radiation with the solvent (water). One can form the same free radicals as in conditions of oxidative stress ((•)OH, O2(•)(-), NO2(•)…). Moreover, the quantity of reactive oxygen (ROS) or nitrogen (RNS) species formed in the irradiated medium can be calculated knowing the dose and the radiation chemical yield, G, thus this method is quantitative. The use of the method of radiolysis has provided a wealth of data, especially about the kinetics of the oxidation by various free radicals and their mechanisms, the identification of transients formed, their lifetimes and the possibility to repair them by the so-called antioxidants. In this review we have collected the most recent data about protein oxidation that might be useful to a proteomic approach. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

Methionine Residue Acts as a Prooxidant in the •OH-Induced Oxidation of Enkephalins

Enkephalins are bioactive pentapeptides (Tyr-Gly-Gly-Phe-Leu (Leu-enk) and Tyr-Gly-Gly-Phe-Met (Met-enk)) produced while an organism is under mental and/or physical stress. In the course of their biological action they are exposed to reactive oxygen and nitrogen species. We have reinvestigated the reactions of (•)OH radicals toward these peptides in order to elucidate the oxidation mechanisms and the final products. Nanosecond pulse radiolysis was used to obtain the spectra of the reaction intermediates and their kinetics. Additional insight into details of the oxidation mechanism was gained by identification of main final products by means of UV-vis spectrophotometry, HPLC coupled with fluorescence spectroscopy, and mass spectrometry. The key processes are different in both peptides. In Leu-enk, the first step is an (•)OH radical addition to the aromatic rings of Tyr and Phe residues that leads to hydroxylated residues, dihydroxyphenylalanine (DOPA) from Tyr and tyrosine isomers from Phe, respectively. In Met-enk, these processes are less important, an additional target being the sulfur atom of the methionine residue. Depending on pH either an OH-adduct (hydroxysulfuranyl radical) or a sulfur radical cation undergo intramolecular electron transfer with Tyr residue resulting in a repair of Met and oxidation of Tyr to tyrosyl radicals and a final formation of dityrosine. At low pH, the OH-adducts to Tyr residue are precursors of tyrosyl radicals and dityrosine. Thus, the final products coming from oxidation of the Tyr residue depend strongly on the neighboring residues and the pH.

Photo-oxidation of Methionine-containing Peptides by the 4-Carboxybenzophenone Triplet State in Aqueous Solution. Competition Between Intramolecular Two-centered Three-electron Bonded (S∴S)+ and (S∴N)+ Formation¶

Abstract Quantum yields for the formation of transients were measured following the quenching of triplet 4-carboxybenzophenone (3CB*) by methionine-containing peptides in aqueous solutions. Ketyl radicals (CBH·), ketyl radical anions (CB·−) and various sulfur radical cations were identified following the triplet-quenching events. The presence of these intermediates indicated that the triplet-quenching mechanism can be characterized as mainly electron-transfer in nature. The quenching rate constants were of the order of 2 × 109 M−1 s−1. There were small, but significant, differences in the triplet-quenching rate constants, and these trends indicate the existence of multiple sulfur targets in the quenchers. The absorption of the transient products was followed in detail by using spectral-resolution analysis. From the absorption data, quantum yields were estimated for the formation of the various transients. There were differences found in the yields of the transient products between the experiments, where the quenchers were the “mixed” stereoisomers of methionylmethionine (l,d and d,l) and experiments where the quenchers were l,l and d,d stereoisomers. Triplet-quenching data from several other methionine-containing small oligopeptides were analyzed in an analogous manner. Systematic variations were observed, and these patterns were discussed in terms of competitive donation of protons to the CB·− within the charge-transfer complex. The competition was between protons on carbons adjacent to the sulfur-radical center and protons on the protonated amino groups of the radical cation. In addition, there was a competition between the two intramolecular two-centered, three-electron bonded species (S∴S)+ and (S∴N)+ that play roles in the secondary kinetics.

Decarboxylation mechanism of the n-terminal glutamyl moiety in γ-glutamic acid and methionine containing peptides

Abstract The reaction of hydroxyl radicals with γ-glutamyl-methionine and γ-glutamyl-glycyl-methionyl-glycine at neutral pH results in similar N-terminal decarboxylation efficiency. The underlying mechanism involves an intramolecular proton transfer from the protonated N-terminal amino group of the glutamyl moiety to an initially formed hydroxy sulphuranyl radical at the methionine residue. This process leads to the formation of a three-electron bonded [> S∴NH 2 ] + - peptide intermediate subsequently decomposing into CO2 and an α-amino radical of the structure H2NCH.CH2CH2C(O)NH- peptide. This radical has been identified via reduction of a moderately good electron acceptor such as p-nitroacetophenone (PNAP). The arrangement within a sterically favourable 5-membered ring, as observed with methionine, is not a necessary prerequisite for the formation of [> S∴NH 2 ] + - type intermediate. Mechanistically, the formation of CO2 and an α-amino radical suggests the occurrence of an intramolecular electron transfer from the carboxylate group to the electron-deficient center at the nitrogen within the S∴N-bond followed by homolytic bond breakage of the carbon-carboxylate bond. The decarboxylation benefits in particular from stabilization of the arising carbon-centered radical by a free lone pair from the α-amino group. This process seems to occur well in larger peptide structures provided they contain an N-terminal carboxyl group α to an amino function and they are flexible enough to allow the protonated amino function to interact with the hydroxyl sulphuranyl radical at the methionine residue.