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Substituted hemins as probes for structure-function relationships in horseradish peroxidase
The Journal of Biological Chemistry
|July 10, 1981
Summary
The protein structure of horseradish peroxidase (HRP) significantly influences the reactivity of its Compound I, dictating its electronic ground state and interaction with hydrogen donors. Substituent positions on the hemin molecule play a crucial role in HRP activity and binding kinetics.
Area of Science:
- Biochemistry
- Enzymology
- Spectroscopy
Background:
- Peroxidases are enzymes that catalyze oxidation-reduction reactions.
- Compound I is a key high-valent intermediate in peroxidase catalysis.
- The electronic and structural properties of Compound I determine enzyme activity.
Purpose of the Study:
- To investigate the electronic ground state of Compound I in peroxidases reconstituted with various hemin derivatives.
- To determine the role of the peroxidase apoprotein structure in influencing Compound I reactivity.
- To examine how hemin substituents interact with the apoprotein and affect enzyme kinetics.
Main Methods:
- Low-temperature visible spectroscopy to analyze Compound I electronic states.
- Reconstitution of peroxidases with modified hemin molecules.
- Comparative analysis of enzyme activity and binding rates with different hemin derivatives.
Main Results:
- Most reconstituted peroxidases exhibited a 2A2u electronic ground state for Compound I.
- Deuterohemin horseradish peroxidase showed a 2A1u ground state, correlating with lack of catalase activity.
- Hemin substituents at the 2- and 4-positions interact sterically with the apoprotein, while 6- and 7-positions show greater sensitivity to structural changes.
Conclusions:
- The peroxidase apoprotein structure is the primary determinant of Compound I reactivity, not solely the electronic ground state.
- Differential interactions between isoelectronic hemins and the apoprotein highlight the importance of substituent positions.
- Subtle changes in hemin side chains (e.g., propionate vs. butyrate) significantly impact enzyme binding and activity, indicating specific protein-hemin interactions.