Identification of a Porphyrin π Cation Radical in Ascorbate Peroxidase Compound I

Abstract

Electron paramagnetic resonance (EPR) spectroscopy has been used to analyze the ascorbate peroxidase Fe3+ resting state and to compare the reaction product between the enzyme and H2O2, compound I, with that of cytochrome c peroxidase. Because ascorbate peroxidase has a Trp residue in the proximal heme pocket at the same location as the Trp 191 compound I free radical in cytochrome c peroxidase [Patterson, W. R., & Poulos, T. L. (1995) Biochemistry 34, 4331-4341], it was anticipated that ascorbate peroxidase compound I might also contain a Trp-centered radical. However, the ascorbate peroxidase compound I EPR spectrum is totally different from that of cytochrome c peroxidase. Immediately after the addition of H2O2, the 7.5 K EPR spectrum of ascorbate peroxidase compound I exhibits an axial resonance extending from g⊥ = 3.27 to g‖ ~ 2 that disappears within 30 s, presumably due to endogenous reduction of compound I. In contrast, cytochrome c peroxidase compound I exhibits a long-lived g #x007E;; 2 signal associated with the Trpl91 cation free-radical [Houseman, A. L. P., et al. (1993) Biochemistry 32, 4430-4443], Recently, the 2 K EPR spectrum of a catalase compound I was found to exhibit a broad signal extending from g⊥ = 3.45 to g‖ ~ 2 and was interpreted as a porphyrin π cation radical [Benecky, M. J., et al. (1993) Biochemistry 32, 11929-11933], On the basis of these comparisons, we conclude that ascorbate peroxidase forms an unstable compound I porphyrin π cation radical, even though it has a Trp residue positioned precisely where the Trpl91 radical is located in cytochrome c peroxidase. © 1995, American Chemical Society. All rights reserved.