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Detection of a tryptophan radical in the reaction of ascorbate peroxidase with hydrogen peroxide
- Hiner, Alexander N. P., MartÃnez, JesÃºs I., Arnao, Marino B., Acosta, Manuel, Turner, Daniel D., Lloyd Raven, Emma, RodrÃguezâLÃ³pez, JosÃ© 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. CompoundâI of rAPX, generated by reaction of rAPX with 1 molar equivalent of H2O2, contains a porphyrin Ïâcation radical. This species is unstable and, in the absence of reducing substrate, decays within 60âs toÂ aÂ second species, compoundâI*, that has a UV/visible spectrum [Î»max (nm)â=â414, 527, 558 and 350 (sh)] similar, but not identical, to those of both horseradish peroxidase compoundâII and cytochromeâc peroxidase compoundâI. Small but systematic differences were observed in the UV/visible spectra of compoundâI* and authentic rAPX compoundâII, generated by reaction of rAPX with 1 molar equivalent H2O2 in the presence of 1 molar equivalent of ascorbate [Î»max (nm)â=â416, 527, 554, 350 (sh) and 628 (sh)]. CompoundâI* decays to give a âferricâlikeâ species (Î»maxâ=â406ânm) that is not spectroscopically identical to ferric rAPX (Î»maxâ=â403ânm) with a first order rate constant, kdecayâ²â=â(2.7âÂ±â0.3)âÃâ10â4âsâ1. Authentic samples of compound II evolve to ferric rAPX [kdecayâ=â(1.1âÂ±â0.2)âÃâ10â3âsâ1]. Low temperature (10âK) EPR spectra are consistent with the formation of a proteinâbased radical, with g values for compoundâI* (gâ¥â=â2.038, gâ¥â=â2.008) close to those previously reported for the Trp191 radical in cytochrome c peroxidase (gâ¥â=â2.037, gâ¥â=â2.005). The EPR spectrum of rAPX compoundâII was essentially silent in the gâ=â2 region. Tryptic digestion of the âferricâlikeâ rAPX followed by RPâHPLC revealed a fragment with a new absorption peak near 330ânm, consistent with the formation of a hydroxylated tryptophan residue. The results show, for the first time, that rAPX can, under certain conditions, form a proteinâbased radical analogous to that found in cytochromeâc peroxidase. The implications of these data are discussed in the wider context of both APX catalysis and radical formation and stability in haem peroxidases.