A SINGLE HISTIDINE IS REQUIRED FOR ACTIVITY OF CYTOCHROME-C PEROXIDASE FROM PARACOCCUS-DENITRIFICANS

The diheme cytochrome c peroxidase from Paracoccus denitrificans was m odified with the histidine-specific reagent diethyl pyrocarbonate. At low excess of reagent, 1 mol of histidine was modified in the oxidized enzyme, and modification was associated with loss of the ability to f orm the active state. With time, the modification reversed, and the ab ility to form the active state was recovered, The agreement between th e spectrophotometric measurement of histidine modification and radioac tive incorporation using a radiolabeled reagent indicated Little modif ication of other amino acids. However, the reversal of histidine modif ication observed spectrophotometrically was not matched by loss of rad ioactivity, and we propose a slow transfer of the ethoxyformyl group t o an unidentified amino acid. The presence of CN- bound to the active peroxidatic site of the enzyme led to complete protection of the essen tial histidine from modification. Limited subtilisin treatment of the native enzyme followed by tryptic digest of the C-terminal fragment (r esidues 251-338) showed that radioactivity was located in a peptide co ntaining a single histidine at position 275. We propose that this cons erved residue, in a highly conserved region, is central to the functio n of the active mixed-valence state.