Role of the proximal ligand in peroxidase catalysis. Crystallographic, kinetic, and spectral studies of cytochrome c peroxidase proximal ligand mutants.
The role of the proximal histidine ligand in peroxidase function was studied by replacing the His side chain in cytochrome c peroxidase with Gln, Glu, or Cys. In addition, a double mutant was prepared where His-175 is converted to Gln and the site of free radical formation in Compound I, Trp-191 (Sivaraja, M., Goodin, D.B., Smith, M., and Hoffman, B. M. (1989) Science 245, 738-740), is converted to Phe. With the exception of the His-175-->Cys mutant, the proximal ligand mutants retain high levels of enzyme activity. Stopped flow studies show that replacing the His ligand with Gln has only a modest effect on the rate of Compound I formation demonstrating that the precise nature of the proximal ligand is not important in achieving a high rate of peroxide O-O bond cleavage. The double mutant, His-175-->Gln/Trp-191-->Phe, also forms Compound I rapidly but the initial product formed is very likely a long-lived porphyrin pi cation radical that slowly converts to a species more closely resembling the heme oxyferryl center of wild type Compound I. The relevance of these studies to the cytochrome c peroxidase-cytochrome c electron transfer system are discussed.
Choudhury
K
K
Department of Molecular Biology & Biochemistry, University of California, Irvine 92717-3900.
Sundaramoorthy
M
M
Hickman
A
A
Yonetani
T
T
Woehl
E
E
Dunn
M F
MF
Poulos
T L
TL
D018360:Crystallography, X-Ray; D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction
D008024:Ligands; D003578:Cytochrome-c Peroxidase