Spectral and kinetic studies on the formation of eosinophil peroxidase compound I and its reaction with halides and thiocyanate

Compound I of peroxidases takes part in both the peroxidation and the halog enation reaction. This study for the first time presents transient kinetic measurements of the formation of compound I of human eosinophil peroxidase (EPO) and its reaction with halides and thiocyanate, using the sequential-m ixing stopped-flow technique. Addition of I equiv of hydrogen peroxide to n ative EPO leads to complete formation of compound I. At pH 7 and 15 degrees C, the apparent second-order rate constant is (4.3 +/- 0.4) x 10(7) M-1 s(- 1). The rate for compound I formation by hypochlorous acid is (5.6 +/- 0.7) x 10(7) M-1 s(-1). EPO compound I is unstable and decays to a stable inter mediate with a compound II-like spectrum. At pH 7, the two-electron reducti on of compound I to the native enzyme by thiocyanate has a second-order rat e constant of (1.0 +/- 0.5) x 10(8) M-1 s(-1). Iodide [(9.3 +/- 0.7) x 10(7 ) M-1 s(-1)] is shown to be a better electron donor than bromide [(1.9 +/- 0.1) x 10(7) M-1 s(-1)], whereas chloride oxidation by EPO compound I is ex tremely slow [(3.1 +/- 0.3) x 10(3) M-1 s(-1)]. The pH dependence studies s uggest that a protonated form of compound I is more competent in oxidizing the anions. The results are discussed in comparison with those of the homol ogous peroxidases myeloperoxidase and lactoperoxidase and with respect to t he role of EPO in host defense and tissue injury.