Plant peroxidases: substrate complexes with mechanistic implications.

Area of Science:

• Biochemistry and structural biology of plant enzymes.
• Enzymology and protein-ligand interactions.

Background:

• Plant peroxidases are enzymes known to bind phenolic compounds.
• Horseradish peroxidase C (HRP C) is a well-studied plant peroxidase.
• Ferulic acid is a common phenolic compound in plant cell walls and a natural substrate.

Purpose of the Study:

• To elucidate the structural basis of phenolic substrate binding and oxidation by plant peroxidases.
• To investigate the role of specific amino acid residues, such as distal arginine, in enzyme function.
• To propose a general mechanism for peroxidase substrate oxidation.

Main Methods:

• Crystallization of horseradish peroxidase C (HRP C) in complex with benzhydroxamic acid.
• Determination of X-ray crystal structures of HRP C complexed with ferulic acid (binary) and ferulic acid with cyanide (ternary) at high resolution (2.0 Å and 1.45 Å).
• Analysis of structural data to understand enzyme-substrate interactions and flexibility.

Main Results:

• Successful crystallization of HRP C with benzhydroxamic acid.
• High-resolution X-ray structures of HRP C:ferulic acid and HRP C:CN(-):ferulic acid complexes were obtained.
• The structures reveal the flexibility of the aromatic-donor-binding site and the critical role of the distal arginine in substrate oxidation and ligand binding.

Conclusions:

• Plant peroxidases exhibit a flexible substrate-binding site.
• The distal arginine residue is crucial for both substrate oxidation and ligand binding in plant peroxidases.
• A general mechanism for peroxidase substrate oxidation, involving compound I and compound II intermediates, is proposed based on structural and biochemical evidence.