Studies of the radical species in compound ES of cytochrome c peroxidase altered by site-directed mutagenesis.
Yeast cytochrome c peroxidase reacts with hydrogen peroxide to form an intermediate, compound ES, in which the heme iron atom is converted to a ferryl function (Fe4+ = O) and a radical center is generated on a reversibly oxidizable amino acid residue of uncertain identity. As methionine-172 is a possible site of this radical, we have constructed specific variants of cytochrome c peroxidase in which methionine-172 is replaced by serine or cysteine. These mutants and the wild-type enzyme have been expressed in Saccharomyces cerevisiae, purified, and crystallized. Both mutant enzymes are fully active. A stable, reversible, peroxide-induced intermediate with optical properties characteristic of compound ES is observed for the three forms of the enzyme. The electron paramagnetic resonance spectrum of this intermediate at 93 K for the serine mutant exhibits the narrow free-radical signal and hyperfine structure observed for the wild-type enzyme. However, a broader component of the signal that is observed for the wild-type enzyme at this temperature is absent from the spectrum observed with the serine mutant. These results demonstrate that the narrow component of the free-radical signal observed at 93 K cannot reside at methionine-172. The absence of the broader component of the signal for the serine mutant may reflect the loss of spin density on methionine or, alternatively, could arise from conformationally induced changes in the properties of the radical. The results are consistent with a heterogeneity of radical species in the ES complex.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine; D003578:Cytochrome-c Peroxidase / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D005800:Genes, Fungal; D007700:Kinetics; D008715:Methionine; D009154:Mutation; D010544:Peroxidases / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012694:Serine; D013329:Structure-Activity Relationship
D005609:Free Radicals; D012694:Serine; D008715:Methionine; D010544:Peroxidases; D003578:Cytochrome-c Peroxidase; D003545:Cysteine