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

Journal Article · Tue Sep 18 00:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00489a042· OSTI ID:5920930

Satterlee, J D ^[1]; Erman, J E ^[2]; Mauro, J M; Kraut, J ^[3]

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1. Univ. of New Mexico, Albuquerque (USA)
2. Northern Illinois Univ., DeKalb (USA)
3. 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), Journal Name: Biochemistry; (USA) Vol. 29:37; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
ALKALI METAL COMPOUNDS
AMINO ACIDS
ARGININE
ASPARTIC ACID
AZOLES
BACTERIA
BARYONS
CARBOXYLIC ACIDS
CYTOCHROME OXIDASE
CYTOCHROMES
ELEMENTARY PARTICLES
ENZYMES
ESCHERICHIA COLI
EUMYCOTA
FERMIONS
FUNGI
HADRONS
HAEM DEHYDROGENASES
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HISTIDINE
HYPERFINE STRUCTURE
IMIDAZOLES
LIGANDS
MAGNETIC RESONANCE
MICROORGANISMS
MUTANTS
NITRATES
NITROGEN COMPOUNDS
NUCLEAR MAGNETIC RESONANCE
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDOREDUCTASES
OXYGEN COMPOUNDS
PEROXIDASES
PH VALUE
PIGMENTS
PLANTS
POTASSIUM COMPOUNDS
POTASSIUM NITRATES
PROTEINS
PROTONS
RESONANCE
YEASTS