Identification of a porphyrin pi cation radical in ascorbate peroxidase compound I.
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 Trp191 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 perpendicular = 3.27 to g parallel approximately 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 approximately 2 signal associated with the Trp191 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 perpendicular = 3.45 to g parallel approximately 2 and was interpreted as a porphyrin pi 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 pi cation radical, even though it has a Trp residue positioned precisely where the Trp191 radical is located in cytochrome c peroxidase.
Patterson
W R
WR
Department of Physiology and Biophysics, University of California, Irvine 92717, USA.
Poulos
T L
TL
Goodin
D B
DB
D060387:Ascorbate Peroxidases; D002412:Cations; D003578:Cytochrome-c Peroxidase / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000737:chemistry; D005609:Free Radicals; D005990:Glycerol / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry*; D011166:Porphyrins / Q000032:analysis* / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
D002412:Cations; D005290:Ferric Compounds; D005609:Free Radicals; D011166:Porphyrins; D014364:Tryptophan; D006861:Hydrogen Peroxide; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D003578:Cytochrome-c Peroxidase; D005990:Glycerol