The functional role of the key residues in the active site of peroxidases.

Peroxidases are haem-containing enzymes which catalyse the reduction of hydrogen peroxide by different organic substrates. Plant, fungal and bacterial peroxidases are evolutionarily related and constitute the plant peroxidase superfamily, which can be divided into three structural classes. Class I, of prokaryotic origin, includes cytochrome c peroxidase (CCP); class I1 contains secretory fungal peroxidases such as Coprinus ciwms peroxidase (CIP); and class 111 comprises secretory plant peroxidases such as horseradish peroxidase (HRP). Although the three classes show only approx. 20% sequence identity, they have conserved residues in the haem cavity, namely the distal Arg and His, and the proximal His hydrogen-bonded with a buried Asp side chain, which are functionally and structurally important [ 1 4 . In addition, the crystal structures of CCP [3], lignin peroxidase [4,5], Arthromyces rumosus peroxidase (ARP) [6] and CIP [7] show that an extended H-bond network connects the proximal to the distal side of the haem cavity through the distal water molecules, the distal Arg, the propionate groups of the haem, and the proximal His H-bonded to the oxygen of an aspartic residue. Resonance Raman (RR) spectroscopy combined with site-directed mutagenesis is a powerful technique with which to elucidate the molecular interactions at the enzyme active site of haemoproteins. In fact, its selectivity and sensitivity allow one to obtain information on the haem prosthetic group which is the active site of the enzymes. The extended aromatic system of the porphyrin ring gives rise to two n + n* transitions at about 400 nm