Comparative proton NMR analysis of wild-type cytochrome c peroxidase from yeast, the recombinant enzyme from Escherichia coli, and an Asp-235 yields Asn-235 mutant
- Univ. of New Mexico, Albuquerque (USA)
- Northern Illinois Univ., DeKalb (USA)
- Univ. of California, San Diego (USA)
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 {yields} 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{sup {minus}}, N{sub 3}{sup {minus}}, and CN{sup {minus}} 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).
- OSTI ID:
- 5920930
- Journal Information:
- Biochemistry; (USA), Vol. 29:37; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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PEROXIDASES
NUCLEAR MAGNETIC RESONANCE
ARGININE
ASPARTIC ACID
CYTOCHROME OXIDASE
CYTOCHROMES
ESCHERICHIA COLI
HISTIDINE
HYPERFINE STRUCTURE
LIGANDS
MUTANTS
PH VALUE
POTASSIUM NITRATES
PROTONS
YEASTS
ALKALI METAL COMPOUNDS
AMINO ACIDS
AZOLES
BACTERIA
BARYONS
CARBOXYLIC ACIDS
ELEMENTARY PARTICLES
ENZYMES
EUMYCOTA
FERMIONS
FUNGI
HADRONS
HAEM DEHYDROGENASES
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
IMIDAZOLES
MAGNETIC RESONANCE
MICROORGANISMS
NITRATES
NITROGEN COMPOUNDS
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDOREDUCTASES
OXYGEN COMPOUNDS
PIGMENTS
PLANTS
POTASSIUM COMPOUNDS
PROTEINS
RESONANCE
550601* - Medicine- Unsealed Radionuclides in Diagnostics