Where is the radical in compound I of cytochrome c peroxidase? Clues from crystallography and mutagenesis.
A difference Fourier map shows small structural perturbations on oxidation of cytochrome c peroxidase (CCP) to its semi-stable intermediate, compound I. Least-squares refinement of both CCP and compound I quantifies these perturbations and suggests that the radical site may be on the distal side of the heme, since that is where most of the small movements occur. Several engineered mutants of CCP were created in an attempt to assess the function of various side chains, among them Trp-51----Phe, Trp-191----Phe and His-181----Gly. X-ray structures of the mutant CCP's confirm that only minimal changes are caused by these substitutions. Preliminary examination of the mutants' kinetic properties show that Trp-51 is not the radical site; that Trp-191 has an important enzymic function; and that His-181 is not essential for electron transfer, but probably has some more indirect role. The locus of the radical in compound I, however, remains to be established.
Edwards
S L
SL
Department of Chemistry, University of California, San Diego, La Jolla.
Mauro
J M
JM
Fishel
L A
LA
Wang
J M
JM
Miller
M A
MA
Xuong
N H
NH
Kraut
J
J
D001665:Binding Sites; D003578:Cytochrome-c Peroxidase / Q000235:genetics / Q000378:metabolism*; D005609:Free Radicals; D005998:Glycine; D006639:Histidine; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010544:Peroxidases / Q000378:metabolism*; D010649:Phenylalanine; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014364:Tryptophan
D005609:Free Radicals; D010649:Phenylalanine; D006639:Histidine; D014364:Tryptophan; D010544:Peroxidases; D003578:Cytochrome-c Peroxidase; D005998:Glycine