Superoxide-mediated Formation of Tyrosine Hydroperoxides and Methionine Sulfoxide in Peptides through Radical Addition and Intramolecular Oxygen Transfer

The chemistry underlying superoxide toxicity is not fully understood. A potential mechanism for superoxide-mediated injury involves addition to tyrosyl radicals, to give peptide or protein hydroperoxides. The rate constant for the reaction of tyrosyl radicals with superoxide is higher than for dimerization, but the efficiency of superoxide addition to peptides depends on the position of the Tyr residue. We have examined the requirements for superoxide addition and structurally characterized the products for a range of tyrosyl peptides exposed to a peroxidase/ system. These included enkephalins as examples of the numerous proteins and physiological peptides with N-terminal tyrosines. The importance of amino groups in promoting hydroperoxide formation and effect of methionine residues on the reaction were investigated. When tyrosine was N-terminal, the major products were hydroperoxides that had undergone cyclization through conjugate addition of the terminal amine. With non-N-terminal tyrosine, electron transfer from to the peptide radical prevailed. Peptides containing methionine revealed a novel and efficient intramolecular oxygen transfer mechanism from an initial tyrosine hydroperoxide to give a dioxygenated derivative with one oxygen on the tyrosine and the other forming methionine sulfoxide. Exogenous amines promoted hydroperoxide formation on tyrosyl peptides lacking a terminal amine, without forming an adduct. These findings, plus the high hydroperoxide yields with N-terminal tyrosine, can be explained by a mechanism in which hydrogen bonding of to the amine increases is oxidizing potential and alters its reactivity. If this amine effect occurred more generally, it could increase the biological reactivity of and have major implications.Free radical-mediated oxidative damage occurs in numerous diseases and is thought to contribute to the aging process. The primary radical generated by the reduction of oxygen is superoxide (), a relatively benign radical that nevertheless must be removed by superoxide dismutases (SODs)² for an organism to survive in an aerobic environment (1). A number of potentially damaging reactions of have been identified (1–4). One of these, which has received relatively little attention, is the addition of to other radicals to form hydroperoxides (5, 6). This reaction has been shown to occur readily with tyrosine and Tyr-containing dipeptides, resulting in the formation of tyrosine hydroperoxides (5–7). Hydroperoxides are potentially damaging reactive oxygen species. Formation on proteins can result in detrimental structural and functional changes (8). Protein hydroperoxides are also oxidants that can injure other biomolecules.Tyrosyl radicals are generated in many physiological situations and proteins are major targets for reactive oxidants (9). In proteins exposed to free radicals, regardless of the initial site of attack, the resultant radical commonly localizes to Tyr (10–13). Tyrosyl radicals are also produced from tyrosyl peptides through the action of peroxidases such as myeloperoxidase, and are generated during the catalytic cycle of enzymes such as ribonucleotide reductase and cyclooxygenase (14). Tyrosyl radicals undergo a variety of subsequent reactions. They readily dimerize to form dityrosine, which has been well documented as a product of oxidative injury (15, 16). Another oxidative biomarker, nitrotyrosine, is also formed via tyrosyl radicals (4, 15, 17). However, one of their most favored reactions is with (5, 7, 18, 19). The reaction has a rate constant several times higher than that for dimerization (7, 20) and is favored over dityrosine formation in situations where both tyrosyl and radicals are generated (7, 20).The reaction of with phenoxyl radicals results in either repair of the parent phenol (reaction 2, Fig. 1b) or addition to form a hydroperoxide (reaction 3). With tyrosine, most of the reacts by addition (7, 20). The structure of tyrosine hydroperoxide has not been determined directly but inferred from NMR studies of the corresponding monoxide derivative formed by slow decomposition (7). These were shown to be bicyclic compounds formed by conjugate addition of the amino group to the phenol ring (HOHICA, designated I and named in full in Fig. 1b, proposed to arise from reactions 5 and 6).Open in a separate windowFIGURE 1.a, experimental system used for the generation of superoxide and tyrosyl (TyrO^·) radicals. b, proposed mechanism for the formation and decomposition of tyrosine hydroperoxide derivatives. R and R′ represent OH and H, respectively, for Tyr, or amino acid residue(s) for the peptides. Reaction 1 shows peroxidase-mediated formation of Tyr radicals, which can either dimerize (not shown) or react with by electron transfer (reaction 2) or addition (reaction 3). Addition results in the formation of hydroperoxides (o- and p-isomers, only the o-isomer shown) that may exist transiently and decompose to release ¹O₂ (reaction 4) or form a stable species that can undergo conjugate addition of the terminal amino group are shown (when R′= H, reaction 5). An equivalent reaction is proposed for non-N-terminal Tyr (R′= amino acid residue) in which conjugation involves the amide nitrogen. Hydrolysis of the hydroperoxides that are modified by conjugate addition gives the corresponding hydroxide derivatives (I, 3a-hydroxy-6-oxo-2,3,3a,6,7,7a-hexahydro-1H-indol-2-carboxylic acid or HOHICA) in reaction 6. c, possible alternative hydrolysis products (mono-oxygenated derivatives). II, 3,4-dihydroxyphenylalanine derivatives from the o-isomer; III, 4-alanyl-4-hydroxy-cyclohexadienone (HACHD) derivatives from the p-isomer.Hydroperoxide formation has been observed with peptides but only when tyrosine is N-terminal or the reaction is promoted by amino compounds (5). The amine effect has implications for hydroperoxide formation on proteins, but the mechanism is not understood. It has also been postulated that the repair mechanism involves singlet oxygen release from an intermediate (reaction 4) rather than electron transfer (reaction 2) (18), but this has not been studied experimentally.The objectives of this investigation were to determine the structures of the hydroperoxide and any other superoxide addition products, and to understand the mechanism of formation, using a range of synthetic and physiological tyrosyl peptides. These include the opioids Leu- and Met-Enkephalin (Leu-Enk, YGGFL; and Met-Enk, YGGFM, respectively) and Endomorphin 2 (Endo2, YPFF). The opioids have a free N-terminal Tyr that is essential for activity and are potential physiological targets for inactivation by addition. We also investigated whether the presence of a Met residue (as in Met-Enk) influences Tyr-hydroperoxide formation on the peptide and whether addition results in the formation of methionine sulfoxide. If so, this could be a physiological mechanism for production of methionine sulfoxide, which is one of the most prevalent products of oxidative stress (21, 22).Peptides were exposed to a xanthine oxidase (XO) system to generate and hydrogen peroxide (H₂O₂) plus horseradish peroxidase (HRP) to catalyze the reaction of H₂O₂ with the peptide to give the tyrosyl radical (Fig. 1a). Products were analyzed using a general hydroperoxide assay (Fe²⁺/xylenol orange or FOX assay) and by liquid chromatography/electrospray mass spectrometry (LC/MS). We have obtained structural information on the hydroperoxides, identified a mechanism of rapid intramolecular oxidation of Met residues via a hydroperoxide intermediate, and provide an explanation for why amino groups facilitate the addition of to the tyrosyl radical.