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Stopped-flow kinetic study of the peroxidase reactions of mangano-microperoxidase-8
Hui-Chun Yeh1, Chia-Huei Yu, Jinn-Shyan Wang
1Department of Chemistry, National Taiwan University, Taipei, 106, Taiwan, ROC.
Abstract:
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.
Insights
Mangano microperoxidase 8 (Mn(III)-MP8) kinetics were studied. This peroxidase reaction intermediate formation and substrate reactivity were determined, showing pH-dependent activity and correlation with reduction potential.
Area of Science:
- Biochemistry
- Chemical Kinetics
Background:
- Peroxidase-type reactions are crucial in biological systems.
- Microperoxidases are valuable models for studying enzyme mechanisms.
Purpose of the Study:
- To investigate the reaction kinetics of mangano microperoxidase 8 (Mn(III)-MP8).
- To characterize the intermediate formed during Mn(III)-MP8 oxidation and its subsequent reactions with various substrates.
Main Methods:
- Utilized time-resolved and single-wavelength stopped-flow techniques.
- Studied intermediate formation and substrate reaction kinetics separately.
- Analyzed pH-dependent effects and solvent isotope effects.
Main Results:
- Oxidation of Mn(III)-MP8 by H2O2 at pH 10.7 formed an intermediate (1) with a rate constant of 2.9 x 10^4 M^-1 s^-1.
- Intermediate formation showed no deuterium solvent isotope effect, suggesting homolytic cleavage.
- Substrate reactivity varied over two orders of magnitude, correlating with reduction potential and decreasing sharply with increasing pH.
Conclusions:
- Mn(III)-MP8 exhibits pH-dependent peroxidase-type activity.
- Substrate reactivity is influenced by reduction potential and pH.
- Mn(III)-MP8 reactivity is generally lower than Fe(III)-MP8 for similar substrates.