Essential histidine in bacterial cytochrome c peroxidases

Abstract

The cytochrome c peroxidase from the bacterium Pciracoccus denitrificans is a relative of the extensively characterised enzyme from Pseudomonas aeruginosa.

This study investigates the role of an essential histidine residue in the enzyme mechanism of bacterial peroxidases.

Cytochrome c peroxidase from Paracoccus denitrificans was modified with the histidine-specific reagent diethylpyrocarbonate. The reaction can be followed spectroscopically and, at low excess of reagent, one mol of histidine was modified in the oxidised enzyme. The agreement between the spectrophotometric measurement of histidine modification and radioactive incorporation using a radiolabeled reagent indicated little modification of other amino acids. Modification of this easily modifiable histidine was associated with loss of the enzyme's ability to form the active state. With time, the modification reversed and the ability to form the active mixed valence state was recovered. However the reversal of histidine modification observed spectrophotometrically was not matched by loss of radioactivity and a slow transfer of the ethoxyformyl group to another amino acid is proposed. The presence of CN" bound to the active peroxidatic site of the enzyme completely protected the essential histidine from modification.

In its active form cytochrome c peroxidase is a dimer, with Ca2+ situated at the interface between the two monomers. Under conditions where the dimer is the dominant species modification of only 0.5 mol histidine abolishes enzyme activity

Limited subtilisin treatment of the native enzyme resulted in cleavage at a single peptide bond. Although the two fragments remain tightly associated, the cleaved enzyme is inactive. Modification with radiolabeled diethylpyrocarbonate and subsequent subtilisin treatment, followed by tryptic digestion of a 9k fragment, showed that radioactivity was located in a peptide containing a single histidine 275.

With the benefit of four homologous sequences and the use of secondary structure prediction analysis we can determine that histidine 275 is indeed conserved in the four sequences and is preceded by a remarkably unvaried a-helical region suggestive of functional importance. It is proposed that this conserved residue acts as both a catalytic active site residue and a conduit for intermolecular electron transfer in the active mixed-valence high spin-state.