title: Detection of a tryptophan radical in the reaction of ascorbate peroxidase with hydrogen peroxide.
abstract: The reactivity of recombinant pea cytosolic ascorbate peroxidase (rAPX) towards H2O2, the nature of the intermediates and the products of the reaction have been examined using UV/visible and EPR spectroscopies together with HPLC. Compound I of rAPX, generated by reaction of rAPX with 1 molar equivalent of H2O2, contains a porphyrin pi-cation radical. This species is unstable and, in the absence of reducing substrate, decays within 60 s to a second species, compound I*, that has a UV/visible spectrum [lambda(max) (nm) = 414, 527, 558 and 350 (sh)] similar, but not identical, to those of both horseradish peroxidase compound II and cytochrome c peroxidase compound I. Small but systematic differences were observed in the UV/visible spectra of compound I* and authentic rAPX compound II, generated by reaction of rAPX with 1 molar equivalent H2O2 in the presence of 1 molar equivalent of ascorbate [lambda(max) (nm) = 416, 527, 554, 350 (sh) and 628 (sh)]. Compound I* decays to give a 'ferric-like' species (lambda(max) = 406 nm) that is not spectroscopically identical to ferric rAPX (lambda(max) = 403 nm) with a first order rate constant, k(decay)' = (2.7 +/- 0.3) x 10(-4) s(-1). Authentic samples of compound II evolve to ferric rAPX [k(decay) = (1.1 +/- 0.2) x 10(-3) s(-1)]. Low temperature (10 K) EPR spectra are consistent with the formation of a protein-based radical, with g values for compound I* (g parallel = 2.038, g perpendicular = 2.008) close to those previously reported for the Trp191 radical in cytochrome c peroxidase (g parallel = 2.037, g perpendicular = 2.005). The EPR spectrum of rAPX compound II was essentially silent in the g = 2 region. Tryptic digestion of the 'ferric-like' rAPX followed by RP-HPLC revealed a fragment with a new absorption peak near 330 nm, consistent with the formation of a hydroxylated tryptophan residue. The results show, for the first time, that rAPX can, under certain conditions, form a protein-based radical analogous to that found in cytochrome c peroxidase. The implications of these data are discussed in the wider context of both APX catalysis and radical formation and stability in haem peroxidases.
authors.affiliation: Departamento de Biología Vegetal (Fisiología Vegetal), Facultad de Biología, Universidad de Murcia, Spain.
authors.forename: A N
authors.initials: AN
authors.lastname: Hiner
authors.forename: J I
authors.initials: JI
authors.lastname: Martínez
authors.forename: M B
authors.initials: MB
authors.lastname: Arnao
authors.forename: M
authors.initials: M
authors.lastname: Acosta
authors.forename: D D
authors.initials: DD
authors.lastname: Turner
authors.forename: E
authors.initials: E
authors.lastname: Lloyd Raven
authors.forename: J N
authors.initials: JN
authors.lastname: Rodríguez-López
mesh_terms: D060387:Ascorbate Peroxidases; D002851:Chromatography, High Pressure Liquid; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology; D014364:Tryptophan / Q000737:chemistry*; D014466:Ultraviolet Rays
chemical_list: D005609:Free Radicals; D011994:Recombinant Proteins; D014364:Tryptophan; D006861:Hydrogen Peroxide; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D014357:Trypsin