Yeast cytochrome c peroxidase: mutagenesis and expression in Escherichia coli show tryptophan-51 is not the radical site in compound I

Journal Article · Tue Jan 27 04:00:00 EST 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00376a004· OSTI ID:6524366

Fishel, L A; Villafranca, J E; Mauro, J M; Kraut, J

Using oligonucleotide-directed site-specific mutagenesis, they have constructed a system for the mutation and expression of yeast cytochrome c peroxidase (CCP, EC 1.11.1.5) in Escherichia coli and applied it to test the hypothesis that Trp-51 is the locus of the free radical observed in compound I of CCP. The system was created by substituting a CCP gene modified by site-directed mutagenesis, CCP(MI), for the fol gene in a vector previously used for mutagenesis and overexpression of dihydrofolate reductase. E. coli transformed with the resulting plasmid produced the CCP(MI) enzyme in large quantities, more than 15 mg/L of cell culture, of which 10% is holo- and 90% is apo-CCP(MI). The apoenzyme was easily converted to holoenzyme by the addition of bovine hemin. Purified CCP(MI) has the same catalytic activity and spectra as bakers' yeast CCP. A mutation has been made in CCP(MI), Trp-51 to Phe. The Phe-51 mutant protein CCP(MI,F51) is fully active, and the electron paramagnetic resonance (EPR) spectrum, at 89 K, of its oxidized intermediate, compound I, displays a strong sharp resonance at g = 2.004, which is very similar to the signal observed for compound I of both bakers' yeast CCP and CCP(MI). However, UV-visible and EPR spectroscopy revealed that the half-life of CCP(MI,F51) compound I at 23 /sup 0/C is only 1.4% of that observed for the compound I forms of CCP(MI) or bakers' yeast CCP. Thus, Trp-51 is not necessary for the formation of the free radical observed in compound I but appears to exert a significant influence on its stability.

• Research Organization: Univ. of California, San Diego
• OSTI ID: 6524366
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:2; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
ABSORPTION SPECTROSCOPY
AMINO ACIDS
AROMATICS
AZAARENES
AZOLES
BACTERIA
BACTERIOPHAGES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CYTOCHROME OXIDASE
CYTOCHROMES
DAYS LIVING RADIOISOTOPES
DNA SEQUENCING
ELECTRON SPECTRA
ELECTRON SPIN RESONANCE
ENZYMES
ESCHERICHIA COLI
EVEN-ODD NUCLEI
FUNGI
HAEM DEHYDROGENASES
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
INDOLES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
MUTAGENESIS
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDOREDUCTASES
PARASITES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PIGMENTS
PLANTS
PROTEINS
PYRROLES
RADIOISOTOPES
RESONANCE
SACCHAROMYCES
SACCHAROMYCES CEREVISIAE
SPECTRA
SPECTROSCOPY
STRUCTURAL CHEMICAL ANALYSIS
SULFUR 35
SULFUR ISOTOPES
TRACER TECHNIQUES
TRYPTOPHAN
ULTRAVIOLET SPECTRA
VIRUSES
YEASTS