Methionine oxidation with hydrogen peroxide

The thioether side chains of methionine units in proteins can be oxidized with hydrogen peroxide to the corresponding sulfones (Eq. 3-48). They can also be alkylated, e.g., by CH3I to form R—S+(CH3)2. 

In order to prevent S-alkylation in the repetitive acid-cleavage steps required in a multistep peptide synthesis, the protection of methionine residues as sulfoxides was proposed by Iselin.f This approach has since been widely used for peptide synthesis in solution and on solid supports.Generally, oxidation of L-methionine yields a mixture of S- and R-sulf-oxides and, depending on the conditions used, even a mixture of the related sulfone. Thereby, at least by oxidation with hydrogen peroxide the R-sulfoxide is formed in a preferred manner (80%), whereas oxidation of L-methionine by tetrachloroauric(III) acid has been reported to produce stereospecifically the 5-sulfoxide diastereoisomer. Isomeric pure Met(O) derivatives are obtained by isolation of the isomers from the 5,/ -sulfoxide mixture, taking advantage of the differences in solubility of the picrate salts.0 ... 

Strong support for this assumption has been presented by Mutt (1964), who showed that cholecystokinin, which contains methionine, in line with the pituitary adrenocorticotropic hormone, the a- and p-melanocyte stimulating hormones and the parathyroid hormone can be oxidized by hydrogen peroxide with complete loss of activity and reactivated with cysteine to practically full strength. The pancreozymin activity simultaneously underwent the same changes. Under similar conditions the pure secretin, which does not contain methionine, did not show any change in activity. 

There is some information concerning the reaction of ozone with chemicals under aqueous conditions. The information available suggests that double-bond cleavage takes place, just as it does under nonaqueous conditions, except that ozonides are not formed. Instead, the zwitterionk intermediate reacts with water, producing an aldehyde and hydrogen peroxide. In addition to double-bond cleavage, a number of other oxidations are possible. Mudd et showed that the susceptibility of amino acids is in the order cysteine, tryptophan, methionine. 

The sulfur atom of methionine residues may be modified by formation of sulfonium salts or by oxidation to sulfoxides or the sulfone. The cyanosulfonium salt is not particularly useful for chemical modification studies because of the tendency for cyclization and chain cleavage (129). This fact, of course, makes it very useful in sequence work. Normally, the methionine residues of RNase can only be modified after denaturation of the protein, i.e., in acid pH, urea, detergents, etc. On treatment with iodoacetate or hydrogen peroxide, derivatives with more than one sulfonium or sulfoxide group did not form active enzymes on removal of the denaturing agent (130) [see, however, Jori et al. (131)]. There was an indication of some active monosubstituted derivatives (130, 132). 

Peroxynitrite is a nonspecific oxidant that reacts with all classes of biomolecules depleting low-molecular-weight antioxidants, initiating lipid peroxidation, damaging nucleic acids and proteins. Its reactions are much slower than those of the hydroxyl radical but are faster than those of hydrogen peroxide. Comparison of peroxynitrite reactivity with various amino acid residues of human serum albumin have shown that cysteine, methionine, and tryptophan are the most reactive... 

This is usually isolated from yeast, and has a molecular weight of 53 000 with one heme b. It catalyzes the oxidation of ferrocytochrome c by hydrogen peroxide. It is the first peroxidase for which the structure has been determined. The imidazole axial ligand is His-174, while Arg-48, Trp-51 and His-52 provide distal catalytic groups. The mechanism involves nucleophilic attack of peroxide on the Fe, loss of the ROOH proton to the imidazole of His-52, and transfer of this proton to the leaving RO group. Compound I of cytochrome c peroxidase is red, and differs from HRPI in that the additional oxidizing equivalent is on a protein residue. ESR and ENDOR spectra have been interpreted in terms of a methionine-centred free radical. One possibility is that a ferryl porphyrin cation radical is formed with cytochrome c peroxidase (it is attractive to assume that this would be common to all peroxidases), but that cytochrome c peroxidase has a readily oxidizable substrate which reduces the porphyrin radical. ... 

The course of the reaction of hydrogen peroxide and several other oxidizing reagents with proteins has been reviewed by Means and Feeney (16). The vulnerable protein sites include those of cysteine and methionine, and in certain instances cystine, tryptophan, and tyrosine. These reactions are of interest to the protein biochemist concerned with protein structure and bioactivity, yet the fundamental concern here is whether oxidation results in beneficial alteration of properties for food and whether such treatment results in diminished nutritive value. 

Some microorganisms in culture show methionine-dependent ethylene formation. In studies with Escherichia coli, 2-oxo-4-methylthiobutyrate (KMB) produced from methionine by transamination was suggested as the precursor of ethylene [19], and subsequently a cell-free system which produced ethylene from KMB in the presence of NAD(P)H, EDTA-Fe and oxygen was established [20]. An enzyme which catalysed a similar ethylene-forming activity was purified from Cryptococcus albidus [15]. The purified enzyme of molecular mass 62 kDa turned out to be NADH EDTA-Fe oxidoreductase. The proposed mechanism involves reduction of EDTA-Fe to EDTA-Fe by the enzyme, reduction of oxygen to superoxide by EDTA-Fe, of hydrogen peroxide to hydroxyl radical, and oxidation of KMB by hydroxyl radical to ethylene. However, an extensive physiological evaluation of this enzyme must be done before it can... 

---