A single histidine is required for activity of cytochrome c peroxidase from Paracoccus denitrificans.
The diheme cytochrome c peroxidase from Paracoccus denitrificans was modified 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 form the active state. With time, the modification reversed, and the ability to form the active state was recovered. The agreement between the spectrophotometric measurement of histidine modification and radioactive incorporation using a radiolabeled reagent indicated little modification of other amino acids. However, the reversal of histidine modification observed spectrophotometrically was not matched by loss of radioactivity, and we propose a slow transfer of the ethoxyformyl group to an unidentified amino acid. The presence of CN- bound to the active peroxidatic site of the enzyme led to complete protection of the essential histidine from modification. Limited subtilisin treatment of the native enzyme followed by tryptic digest of the C-terminal fragment (residues 251-338) showed that radioactivity was located in a peptide containing a single histidine at position 275. We propose that this conserved residue, in a highly conserved region, is central to the function of the active mixed-valence state.
McGinnity
D F
DF
Department of Preclinical Veterinary Sciences, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Summerhall, United Kingdom.
Devreese
B
B
Prazeres
S
S
Van Beeumen
J
J
Moura
I
I
Moura
J J
JJ
Pettigrew
G W
GW
D000595:Amino Acid Sequence; D001665:Binding Sites; D002250:Carbon Radioisotopes; D002850:Chromatography, Gel; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D003486:Cyanides; D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000378:metabolism / Q000494:pharmacology*; D004578:Electron Spin Resonance Spectroscopy; D006639:Histidine; D007700:Kinetics; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet; D013381:Subtilisins; D014357:Trypsin
D002250:Carbon Radioisotopes; D003486:Cyanides; D010446:Peptide Fragments; D006639:Histidine; D003578:Cytochrome-c Peroxidase; D013381:Subtilisins; D014357:Trypsin; D004047:Diethyl Pyrocarbonate