Comparative proton NMR analysis of wild-type cytochrome c peroxidase from yeast, the recombinant enzyme from Escherichia coli, and an Asp-235----Asn-235 mutant.
Proton NMR spectra of cytochrome c peroxidase (CcP) isolated from yeast (wild type) and two Escherichia coli expressed proteins, the parent expressed protein [CcP(MI)] and the site-directed mutant CcP(MI,D235N) (Asp-235----Asn-235), have been examined. At neutral pH and in the presence of only potassium phosphate buffer and potassium nitrate, wild-type Ccp and CcP(MI) demonstrate nearly identical spectra corresponding to normal (i.e., "unaged") high-spin ferric peroxidase. In contrast, the mutant protein displays a spectrum characteristic of a low-spin form, probably a result of hydroxide ligation. Asp-235 is hydrogen-bonded to the proximal heme ligand, His-175. Changing Asp-235 to Asn results in alteration of the pK for formation of the basic form of CcP. Thus, changes in proximal side structure mediate the chemistry of the distal ligand binding site. All three proteins bind F-, N3-, and CN- ions, although the affinity of the mutant protein (D235N) for fluoride ion appears to be much higher than that of the other two proteins. Analysis of proton NMR spectra of the cyanide ligated forms leads to the conclusion that the mutant protein (D235N) possesses a more neutral proximal histidine imidazole ring than does either wild-type CcP or CcP(MI). It confirms that an important feature of the cytochrome c peroxidase structure is at least partial, and probably full, imidazolate character for the proximal histidine (His-175).
Satterlee
J D
JD
Department of Chemistry, Washington State University, Pullman 99164-4630.
Erman
J E
JE
Mauro
J M
JM
Kraut
J
J
D001216:Asparagine; D001224:Aspartic Acid; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006418:Heme; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D006418:Heme; D001216:Asparagine; D003578:Cytochrome-c Peroxidase