Stopped-flow kinetic study of the peroxidase reactions of mangano-microperoxidase-8.
We have investigated the kinetics for the peroxidase-type reaction of mangano microperoxidase 8 (Mn(III)-MP8) by the time-resolved and single-wavelength stopped-flow technique. The formation of intermediate and its subsequent reaction with substrates were studied separately. Oxidation of Mn(III)-MP8 by H2O2 at pH 10.7 yields an intermediate (1) with a rate constant of 2.9 x10(4) M-1 s-1. The formation of 1 exhibits no deuterium solvent isotope effect, favoring the homolytic cleavage of the Mn(III)-MP8 bound hydroperoxide. The rate for the formation of 1 increases sharply as the pH increases and no other intermediate was detected in the entire pH range. Addition of substrate to 1 leads to the regeneration of Mn(III)-MP8. Monitoring the conversion of 1 to Mn(III)-MP8 allows the determination of the substrate reactivity. The substrate reactivity varies by more than two orders of magnitude ranging from 1.04 x 10(6) M-1 s-1 for ascorbic acid to 4.61 x 10(3) M-1s-1 for aniline. It is linearly correlated with the reduction potential for most of the substrates studied, with the easier oxidized species showing greater reactivity. The substrate reactivity drops rapidly as the pH increases. The substrate reactivity at pH 10.7 for the Mn(III)-MP8 system is smaller than that of the corresponding Fe(III)-MP8 system by 2- to 25-fold, depending on the substrate used.
Yeh
Hui-Chun
HC
Department of Chemistry, National Taiwan University, Taipei, 106, Taiwan, ROC.
Yu
Chia-Huei
CH
Wang
Jinn-Shyan
JS
Chen
Suei-Tein
ST
Su
OliverY
O
Lin
Wann-Yin
WY
D000818:Animals; D003903:Deuterium; D004579:Electron Transport / Q000502:physiology; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000378:metabolism*; D009284:Naphthols / Q000378:metabolism*; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity
D009284:Naphthols; C029350:1-naphthol; D008345:Manganese; D003903:Deuterium; D006861:Hydrogen Peroxide; D007501:Iron; D010544:Peroxidases; C021799:microperoxidase