PubAg

Main content area

Detection of a tryptophan radical in the reaction of ascorbate peroxidase with hydrogen peroxide

Author:
Hiner, Alexander N. P., Martínez, Jesús I., Arnao, Marino B., Acosta, Manuel, Turner, Daniel D., Lloyd Raven, Emma, Rodríguez‐López, José Neptuno
Source:
European journal of biochemistry 2001 v.268 no.10 pp. 3091-3098
ISSN:
0014-2956
Subject:
absorption, ascorbate peroxidase, catalytic activity, cytochrome-c peroxidase, digestion, electron paramagnetic resonance spectroscopy, hydrogen peroxide, peas, peroxidase, porphyrins, reversed-phase high performance liquid chromatography, temperature, tryptophan
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 π‐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 [λmax (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 [λmax (nm) = 416, 527, 554, 350 (sh) and 628 (sh)]. Compound I* decays to give a ‘ferric‐like’ species (λmax = 406 nm) that is not spectroscopically identical to ferric rAPX (λmax = 403 nm) with a first order rate constant, kdecay′ = (2.7 ± 0.3) × 10−4 s−1. Authentic samples of compound II evolve to ferric rAPX [kdecay = (1.1 ± 0.2) × 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∥ = 2.038, g⊥ = 2.008) close to those previously reported for the Trp191 radical in cytochrome c peroxidase (g∥ = 2.037, g⊥ = 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.
Agid:
108452