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

Hiner, Alexander N. P., Martínez, Jesús I., Arnao, Marino B., Acosta, Manuel, Turner, Daniel D., Lloyd Raven, Emma, Rodríguez‐López, José Neptuno
European journal of biochemistry 2001 v.268 no.10 pp. 3091-3098
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
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.