Structure of horseradish peroxidase compound I. Kinetic evidence for the incorporation of one oxygen atom from the oxidizing substrate into the enzyme.

The kinetics of the reaction between horseradish peroxidase and p-nitroperbenzoic acid to form compound I have been studied at 25 degrees C in phosphate buffer pH 7.2 and ionic strength of 0.11 M by transient-state and steady-state methods. The second-order rate constant for compound I formation obtained by stopped-flow measurements at 403 nm is (3.7 +/- 0.2) x 10(7) M-1 s-1. For the disappearance of p-nitroperbenzoic acid and appearance of p-nitrobenzoic acid using steady-state kinetics measured at 265 nm the rate constant is (3.0 +/- 0.6) x 10(7) M-1 s-1. The results provide an independent confirmation that one and only one oxygen atom is incorporated from the oxidizing substrate into compound I.