title: Conversion of the proximal histidine ligand to glutamine restores activity to an inactive mutant of cytochrome c peroxidase.
abstract: Using site-directed mutagenesis, a double mutant in yeast cytochrome c peroxidase (CCP) has been constructed where the proximal ligand, His175, has been converted to glutamine and the neighboring Trp191 has been converted to phenylalanine. The refined 2.4-A crystal structure of the double mutant shows that the Gln175 side chain is within coordination distance of the heme iron atom and that Phe191 occupies the same position as Trp191 in the native enzyme with very little rearrangement outside the immediate vicinity of the mutations. Consistent with earlier work, we find that the single mutant, His175-->Gln, is fully active under steady state assay conditions and that as reported earlier (Mauro et al., 1988), the Trp191-->Phe mutant exhibits only < 0.05% activity. However, the double mutant, His175-->Gln/Phe191-->Phe, exhibits 20% wild type activity. Since it is known that the Trp191-->Phe mutant is inactive because it can no longer transfer electrons from ferrocytochrome c, changing the nature of the proximal ligand is able to restore this activity. These results raise interesting questions regarding the mechanism of interprotein electron transfer reactions.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of California, Irvine 92717.
authors.forename: K
authors.initials: K
authors.lastname: Choudhury
authors.forename: M
authors.initials: M
authors.lastname: Sundaramoorthy
authors.forename: J M
authors.initials: JM
authors.lastname: Mauro
authors.forename: T L
authors.initials: TL
authors.lastname: Poulos
mesh_terms: D000595:Amino Acid Sequence; D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005973:Glutamine; D006632:Histamine; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013816:Thermodynamics; D014961:X-Ray Diffraction
chemical_list: D008024:Ligands; D011994:Recombinant Proteins; D005973:Glutamine; D006632:Histamine; D003578:Cytochrome-c Peroxidase