Spectral and kinetic studies on eosinophil peroxidase compounds I and II and their reaction with ascorbate and tyrosine

Eosinophil peroxidase, the major granule protein in eosinophils, is the lea st studied human peroxidase. Here, we have performed spectral and kinetic m easurements to study the nature of eosinophil peroxidase intermediates, com pounds I and II, and their reduction by the endogenous one-electron donors ascorbate and tyrosine using the sequential-mixing stopped-flow technique. We demonstrate that the peroxidase cycle of eosinophil peroxidase involves a ferryl/porphyrin radical compound I and a ferryl compound II. In the abse nce of electron donors, compound I is shown to be transformed to a species with a compound II-like spectrum. In the presence of ascorbate or tyrosine compound I is reduced to compound II with a second-order rate constant of ( 1.0 +/- 0.2) x 10(6) M-1 s(-1) and (3.5 +/- 0.2) x 10(5) M-1 s(-1) respecti vely (pH 7.0, 15 degreesC). Compound II is then reduced by ascorbate and ty rosine to native enzyme with a second-order rate constant of (6.7 +/- 0.06) x 10(3) M-1 s(-1) and (2.7 +/- 0.06) x 10(4) M-1 s(-1), respectively. This study revealed that eosinophil peroxidase compounds I and II are able to r eact with tyrosine and ascorbate via one-electron oxidations and therefore generate monodehydroascorbate and tyrosyl radicals. The relatively fast rat es of the compound I reduction demonstrate that these reactions may take pl ace in vivo and are physiologically relevant. (C) 2001 Elsevier Science B.V . All rights reserved.