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

The reactivity of recombinant pea cytosolic ascorbate peroxidase (rAPX) tow ards H2O2, the nature of the intermediates and the products of the reaction have been examined using UV/visible and EPR spectroscopies together with H PLC. Compound I of rAPX, generated by reaction of rAPX with 1 molar equival ent of H2O2, contains a porphyrin pi -cation radical. This species is unsta ble and, in the absence of reducing substrate, decays within 60 s to a seco nd 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 ascor bate [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 no t 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). Authe ntic 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 th e formation of a protein-based radical, with g values for compound I* (g(<p arallel to>) = 2.038, g(perpendicular to) = 2.008) close to those previousl y reported for the Trp191 radical in cytochrome c peroxidase (g(<parallel t o>) = 2.037, g(perpendicular to) = 2.005). The EPR spectrum of rAPX compoun d 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 absorp tion peak near 330 nm, consistent with the formation of a hydroxylated tryp tophan 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 t he wider context of both APX catalysis and radical formation and stability in haem peroxidases.