IDENTIFICATION OF A PORPHYRIN PI-CATION-RADICAL IN ASCORBATE PEROXIDASE COMPOUND-I

Electron paramagnetic resonance (EPR) spectroscopy has been used to an alyze the ascorbate peroxidase Fe3+ resting state and to compare the r eaction product between the enzyme and H2O2, compound I, with that of cytochrome c peroxidase, Because ascorbate peroxidase has a Trp residu e in the proximal heme pocket at the same location as the Trp191 compo und I free radical in cytochrome c peroxidase [Patterson, W. R., and P oulos, T. L. (1995) Biochemistry 34, 4331-4341], it was anticipated th at ascorbate peroxidase compound I might also contain a Trp-centered r adical. However, the ascorbate peroxidase compound I EPR spectrum is t otally different from that of cytochrome c peroxidase. Immediately aft er the addition of H2O2, the 7.5 K EPR spectrum of ascorbate peroxidas e compound I exhibits an axial resonance extending from g(perpendicula r to) = 3.27 to g(II) similar to 2 that disappears within 30 s, presum ably due to endogenous reduction of compound I. In contrast, cytochrom e c peroxidase compound I exhibits a long-lived g similar to 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 spectru m of a catalase compound I was found to exhibit a broad signal extendi ng from g(perpendicular to) = 3.45 to g(II) similar to 2 and was inter preted 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 porphy rin pi cation radical, even though it has a Trp residue positioned pre cisely where the Trp191 radical is located in cytochrome c peroxidase.