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Effect of modified hemes on the spectral properties and activity of manganese peroxidase
1Biotechnology Center, Utah State University, Logan, Utah, 84322-4705, USA.
Abstract:
Recombinant manganese peroxidase (rMnP), expressed in Escherichia coli as apo-protein, was constituted with Fe(III) protoporphyrin IX, Fe(III) protoporphyrin IX dimethyl ester (DME), Fe(III) deuteroporphyrin (Deut), Fe(III) etioporphyrin III (Etio), and Fe(III) methylpyrrolporphyrin XXI (MPP). The electronic absorption spectra of these hemoproteins were similar to those of native MnP, but absorption maxima were shifted to longer wavelengths in the order of Deut-rMnP, MPP-rMnP, Etio-rMnP, DME-rMnP, and rMnP. All enzymes contained a high-spin, pentacoordinate heme iron as evidenced by the characteristic charge transfer bands in the visible region. The hemoproteins exhibited reduced catalytic activity while maintaining similar substrate Km values compared to native MnP. Compounds I, II, and III were obtained for these hemin-analogue enzymes except for Deut-rMnP. We concluded that the spectral properties of MnP are strongly influenced by porphyrin alpha- and beta-meso edge substituents and manganese oxidation is affected by the gamma-meso edge groups, suggesting a role for the heme propionates in electron transfer during catalysis.
Insights
Recombinant manganese peroxidases (rMnP) reconstituted with various hemin analogues showed altered spectral properties and reduced catalytic activity. Porphyrin substituents influence MnP spectral characteristics and manganese oxidation during catalysis.
Area of Science:
- Biochemistry
- Enzymology
- Protein Chemistry
Background:
- Manganese peroxidase (MnP) is a key enzyme in lignin degradation.
- Understanding MnP's structure-function relationship is crucial for biotechnological applications.
Purpose of the Study:
- To investigate the impact of different hemin analogues on recombinant MnP (rMnP) spectral properties and catalytic activity.
- To elucidate the role of porphyrin substituents in MnP function and electron transfer.
Main Methods:
- Recombinant MnP (apo-protein) was reconstituted with various Fe(III) protoporphyrin IX analogues.
- Electronic absorption spectroscopy was used to analyze hemoprotein spectra.
- Catalytic activity and kinetic parameters (Km) were assessed.
- Enzyme intermediates (Compounds I, II, III) were characterized.
Main Results:
- Reconstituted rMnP exhibited spectral properties similar to native MnP, with shifts in absorption maxima.
- All analogues resulted in high-spin, pentacoordinate heme iron.
- Hemoproteins showed reduced catalytic activity but maintained similar substrate Km values.
- Enzyme intermediates were formed, except for Deut-rMnP.
Conclusions:
- Porphyrin alpha- and beta-meso edge substituents significantly influence MnP spectral properties.
- Gamma-meso edge groups affect manganese oxidation, suggesting a role for heme propionates in electron transfer.
- Hemin analogue substitution provides insights into MnP catalytic mechanisms.