Structure of milk lactoperoxidase. A study using circular dichroism and difference absorption spectroscopy

Area of Science:

• Biochemistry
• Enzymology
• Spectroscopy

Background:

• Milk lactoperoxidase (LPO) is a key enzyme in the innate immune system of milk.
• Understanding the structural and functional properties of LPO is crucial for its applications.

Purpose of the Study:

• To investigate the structural characteristics of milk lactoperoxidase (LPO) and its derivatives using spectroscopic methods.
• To elucidate the nature of the heme pocket and the fifth ligand of the heme iron in LPO.

Main Methods:

• Circular dichroism (CD) spectroscopy in the 200-670 nm region.
• Difference absorption spectroscopy in the far-ultraviolet region.
• Analysis of secondary structure content from CD spectra.

Main Results:

• CD spectra of LPO and its derivatives exhibited split ellipticity bands, indicating a constrained heme pocket.
• Difference absorption spectroscopy confirmed histidine as the fifth ligand of the heme iron.
• Secondary structure analysis revealed a high beta-structure content (65%) in LPO.
• Dithionite reduction of LPO resulted in two forms, suggesting interaction with reduction byproducts near the heme iron.

Conclusions:

• The narrow heme pocket of LPO restricts ligand binding geometry.
• Histidine is confirmed as the proximal ligand to the heme iron.
• LPO possesses a predominantly beta-sheet secondary structure.
• Dithionite reduction introduces complexity, potentially involving byproduct binding near the heme iron.