Stopped-flow kinetic study of the H2O2 oxidation of substrates catalyzed by microperoxidase-8.
We have studied the oxidation of microperoxidase-8 (MP-8) by H2O2 and the subsequent reaction of the intermediates with substrate by stopped-flow experiments. Oxidation of MP-8 by H2O2 gives two intermediates, I and II. The observed rate constant for the formation of I is linearly dependent on [H2O2] and exhibits a bell-shaped dependence on pH with pKa values of 8.90 and 10.60, which are attributed to the deprotonation of MP-bound H2O2 and H2O, respectively. The observed rate constant for the conversion of I to II is independent of [H2O2], but increases sharply at pH>9.0. The predominant forms of the intermediate at pH 7.0 and 10.7 are I and II, respectively. Addition of substrate to the intermediates at pH 9.0 gives rise to three distinct stages, corresponding to the three steps (in decreasing order of rate): I-->II*, II-->MP, and II*-->MP. The rates of these steps are all linearly dependent on the substrate concentration and each individual rate constant has been determined. Substrate reactivity at pH 10.7 covers over two orders of magnitude, ranging from 1.36 x 10(7) M(-1) s(-1) for 1-naphthol to 4.03 x 10(4) M(-1) s(-1) for ferrocyanide. The substrate reactivity is linearly correlated with its reduction potential, indicating that an electron transfer process is involved in the rate-limiting step.
Yeh
H C
HC
Department of Chemistry, National Taiwan University, Taipei 106, Taiwan, ROC.
Wang
J S
JS
Su
Y O
YO
Lin
W Y
WY
D000818:Animals; D005295:Ferrocyanides / Q000737:chemistry; D006736:Horses; D006861:Hydrogen Peroxide / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009284:Naphthols / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D005295:Ferrocyanides; D009284:Naphthols; C029350:1-naphthol; D006861:Hydrogen Peroxide; D010544:Peroxidases; C021799:microperoxidase; C020354:hexacyanoferrate II