Site-specific spin trapping of tyrosine radicals in the oxidation of metmyoglobin by hydrogen peroxide

Abstract

The reaction between metmyoglobin and hydrogen peroxide produces both a ferryl-oxo heme and a globin-centred radical(s) from the two oxidizing equivalents of the hydrogen peroxide. Evidence has been presented for localization of the globin-centred radical on one tryptophan residue and tyrosines 103 and 151. When the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is included in the reaction mixture, a radical adduct has been detected, but the residue at which that adduct is formed has not been determined. Replacement of either tryptophans 7 and 14 or tyrosines 146 and 151 with phenylalanine has no effect on the formation of DMPO adduct in the reaction with hydrogen peroxide. When tyrosine 103 is replaced with phenylalanine, however, only DMPOX, a product of the oxidation of the spin-trap, is detected. Tyrosine-103 is, therefore, the site of radical adduct formation with DMPO. The spin trap 2-methyl-2-nitrosopropane (MNP), however, forms radical adducts with any recombinant sperm whale metmyoglobin that contains either tyrosine 103 or 151. Detailed spectral analysis of the DMPO and MNP radical adducts of isotopically substituted tyrosine radical yield complete structural determinations. The multiple sites of trapping support a model in which the unpaired electron density is spread over a number of residues in the population of metmyoglobin molecules, at least some of which are in equilibrium with each other.

Identifying the site of spin trapping in proteins by a combination of liquid chromatography, ELISA, and off-line tandem mass spectrometry

“…A decrease of conjugation between the ring and the Tyr hydroxyl group associated with the binding of DMPO could conceivably cause such an effect. This explanation is consistent with earlier EPR spin-trapping experiments [31], demonstrating that the Tyr-DMPO radical adduct in the horseradish peroxidase/Tyr/H 2 O 2 system is formed by trapping of the Tyr phenolic oxygen.…”

“…Interestingly, the π-orbitals of Tyr-103 are nearly coplanar with the unsaturated heme system, and this relative orientation has been shown to kinetically favor electron transfer [36]. Thus, our observation that DMPO forms a detectable nitrone adduct with Tyr-103 could have been anticipated and is consistent with previous mass spectrometric (HoMb and SwMb) [14] and site-directed mutagenicity studies (SwMb) [31].…”

Identification of oxidation products and free radicals of tryptophan by mass spectrometry

“…To our knowledge, the detection of tryptophan radicals formed by oxidation reactions, other than in proteins using MS, has not been reported. Some studies reported the observation of tryptophan centered radicals using spin traps and ESR [14,26] or absorbance spectra [27]. The purpose of the present study was to identify oxidation products and radicals formed during oxidation of the tryptophan by hydrogen peroxide, in a Fenton reaction system, in hydrogencarbonate buffer, pH 7.4, similar to physiological conditions.…”

“…The structural information given by this technique is limited, so there is a need for new techniques that provide more information about the origin and location of radical. Recently, it has been shown that mass spectrometry is a suitable technique to detect the stable adducts formed by radicals and spin adducts of DMPO [13][14][15], POBN [16,17], and other [18 -20] spin traps. NMR has also been used in determining the sites of reaction of the hydroxyl radical and in identifying the oxidation products [21,22].…”

Monoamine-dependent Production of Reactive Oxygen Species Catalyzed by Pseudoperoxidase Activity of Human Hemoglobin

“…Similar to Hb, formation of globin-centered radicals with ferryl-oxoheme by H2O2-dependent oxidation of Mb-Fe III was reported. 30) Gunther et al 47) reported that the globin-centered radicals with ferryloxoheme formed after oxidation of Mb-Fe III with H2O2, localizing the radicals at one tryptophan residue and two tyrosine residues.…”