Kinetic studies of the reaction of heme-thiolate enzyme chloroperoxidase with peroxynitrite

The kinetics of the reaction of chloroperoxidase with peroxynitrite was studied under neutral and acidic pH by stopped-flow spectrophotometry. Chloroperoxidase catalyzed peroxynitrite decay with the rate constant, kc, increasing with decreasing pH. The values of kc obtained at pH 5.1, 6.1 and 7.1 were equal to: (1.96 ± 0.03) × 106, (1.63 ± 0.04) × 106 and (0.71 ± 0.01) × 106 M−1 s−1, respectively. Chloroperoxidase was converted to compound II by peroxynitrite with pH-dependent rate constants: (12.3 ± 0.4) × 106 and (3.8 ± 0.3) × 106 M−1 s−1 at pH 5.1 and 7.1, respectively. After most of peroxynitrite had disappeared, the conversion of compound II into the ferric form of chloroperoxidase was observed. The recovery of the native enzyme was completed within 1 s and 5 s at pH 5.1 and 7.1, respectively. The possible reaction mechanisms of the catalytic decomposition of peroxynitrite by chloroperoxidase are discussed.