One-electron oxidative activation of 2-aminofluorene by horseradish peroxidase compounds I and II: spectral and kinetic studies.

Horseradish peroxidase (HRP) catalyzed oxidation of the model carcinogen 2-aminofluorene (2-AF) was studied by rapid scan spectral analysis and stopped flow kinetic measurements. Our rapid scan spectral analysis of the changes in the enzyme oxidation states clearly indicates that the reactions between HRP intermediate compounds and 2-AF are one-electron redox processes, thus a free radical is necessarily the primary product. The reactions were investigated at pH's ranging from 3.40 to 10.00. The pH profiles of rate constants suggested that 2-AF is reactive toward HRP only when it is in its neutral form; one acid-base group of pKa approximately 5 in HRP-I, and one of pKa approximately 8.6 in HRP-II affect the kinetics of HRP catalyzed 2-AF oxidation. The intrinsic second-order rate constants of 2-AF oxidation are (1.1 +/- 0.5) x 10(9) M-1 s-1 and (4.8 +/- 0.4) x 10(8) M-1 s-1 for HRP-I, the two values depending upon the state of protonation of HRP-I, and (1.7 +/- 0.2) x 10(8) M-1 s-1 for HRP-II. We suggest that the rate-controlling step is formation of a substrate cation radical, which is stabilized by the extensive pi-conjugation system of 2-AF. This is supported by the high reactivity of 4-aminobiphenyl, a similar compound, which does not fit the Hammett rho sigma correlations established for other substituted anilines; these other anilines react by hydrogen atom transfer. Thus the extraordinarily rapid reactions between 2-AF and HRP compounds I and II are attributed to the strong aromaticity of 2-AF.