Reactivity of horseradish peroxidase compound II toward substrates: Kinetic evidence for a two-step mechanism

Transient kinetic analysis of biphasic, single turnover data for the reacti on of 2,2'-azino-bis-[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS) with ho rseradish peroxidase (HRPC) compound II demonstrated preequilibrium binding of ABTS (k(+5) = 7.82 x 10(4) M-1 s(-1)) prior to rate-limiting electron t ransfer (k(+6) = 42.1 s(-1)). These data were obtained using a stopped-flow method, which included ascorbate in the reaction medium to maintain a low steady-state concentration of ABTS (pseudo-first-order conditions) and to m inimize absorbance changes in the Soret region due to the accumulation of A BTS cation radicals. A steady-state kinetic analysis of the reaction confir med that the reduction of HRPC compound II by this substrate is rate-limiti ng in the complete peroxidase cycle. The reaction of HRPC with o-diphenols has been investigated using a chronometric method that also included ascorb ate in the assay medium to minimize the effects of nonenzymic reactions inv olving phenol-derived radical products. This enabled the initial rates of o -diphenol oxidation at different hydrogen peroxide and o-diphenol concentra tions to be determined from the lag period induced by the presence of ascor bate. The kinetic analysis resolved the reaction of HRPC compound II with o -diphenols into two steps, initial formation of an enzyme-substrate complex followed by electron transfer from the substrate to the heme. With o-diphe nols that are rapidly oxidized, the heterolytic cleavage of the O-O bond of the heme-bound hydrogen peroxide (k(+2) = 2.17 x 10(3) s(-1)) is rate-limi ting. The size and hydrophobicity of the o-diphenol substrates are correlat ed with their rate of binding to HRPC, while the electron density at the C- 4 hydroxyl group predominantly influences the rate of electron transfer to the heme.