Mechanism of the oxidation of 3,5,3',5'-tetramethylbenzidine by myeloperoxidase determined by transient- and steady-state kinetics.

Earlier investigations of the oxidation of 3,5,3',5'-tetramethylbenzidine (TMB) using horseradish peroxidase and prostaglandin H-synthase have shown the formation of a cation free radical of TMB in equilibrium with a charge-transfer complex, consistent with either a two- or a one-electron initial oxidation. In this work, we exploited the distinct spectroscopic properties of myeloperoxidase and its oxidized intermediates, compounds I and II, to establish two successive one-electron oxidations of TMB. By employing stopped-flow techniques under transient-state and steady-state conditions, we also determined the rate constants for the elementary steps of the myeloperoxidase-catalyzed oxidation of TMB at pH 5.4 and 20 degrees C. The second-order rate constant for compound I formation from the reaction of native enzyme with H2O2 is 2.6 x 10(7) M-1 s-1. Compound I undergoes a one-electron reduction to compound II in the presence of TMB, and the rate constant for this reaction was determined to be (3.6 +/- 0.1) x 10(6) M-1 s-1. The spectral scans show that compound II accumulates in the steady state. The rate constant for compound II reduction to native enzyme by TMB obtained under steady-state conditions is (9.4 +/- 0.6) x 10(5) M-1 s-1. The results are applied to a new, more accurate assay for myeloperoxidase based upon the formation of the charge-transfer complex between TMB and its diimine final product.