X-ray structures of recombinant yeast cytochrome c peroxidase and three heme-cleft mutants prepared by site-directed mutagenesis

Journal Article · Tue Aug 07 04:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00483a003· OSTI ID:5896033

Wang, J; Mauro, J M; Edwards, S L; Oatley, S J; Fishel, L A; Ashford, V A; Xuong, Nguyenhuu; Kraut, J ^[1]

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1. Univ. of California at San Diego, La Jolla (USA)

The 2.2-{angstrom} x-ray structure for CCP(MI), a plasmid-encoded form of Saccharomyces cerevisiae cytochrome c peroxidase (CCP) expressed in Escherichia coli has been solved, together with the structures of three specifically designed single-site heme-cleft mutants. The structure of CCP(MI) was solved by using molecular replacement methods, since its crystals grow differently from the crystals of CCP isolated from bakers' yeast used previously for structural solution. Small distal-side differences between CCP(MI) and bakers' yeast CCP are observed, presumably due to a strain-specific Thr-53 {yields} Ile substitution in CCP(MI). The observation of a vacant sixth coordination site in this structure differs from the results of solution resonance Raman studies, which predict hexacoordinated high-spin iron. The coordination behavior of this W51F mutant is apparently altered in the presence of a precipitating agent, 30% 2-methyl-2,4-pentanediol. A proximal Trp-191 {yields} Phe mutant that has substantially diminished enzyme activity and altered magnetic properties accommodates the substitution by allowing the side chain of Phe-191, together with the segment of backbone to which it is attached, to move toward the heme. This relatively large local perturbation is accompanied by numerous small adjustments resulting in a slight overall compression of the enzyme's proximal domain; however, the iron coordination sphere is essentially unchanged. This structures rules out a major alteration in protein conformation as a reason for the dramatically decreased activity of the W191F mutant. From the alteration of local structure that occurs in this mutant, coupled with the results of preliminary functional studies, the authors infer that Asp-235 exerts influence on the heme iron so as to keep its sixth coordination site vacant, and hence reactive with peroxide substrate, over a wide pH range.

• OSTI ID: 5896033
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:31; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
400102 -- Chemical & Spectral Procedures
550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
AROMATICS
AZAARENES
AZOLES
BACTERIA
CARBOXYLIC ACIDS
COHERENT SCATTERING
CONFORMATIONAL CHANGES
CYTOCHROMES
DIFFRACTION
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
EUMYCOTA
FOURIER ANALYSIS
FUNGI
GENE MUTATIONS
GENETIC ENGINEERING
HEME
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
INDOLES
MICROORGANISMS
MUTAGENESIS
MUTATIONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDOREDUCTASES
PEROXIDASES
PHENYLALANINE
PIGMENTS
PLANTS
PORPHYRINS
PROTEIN ENGINEERING
PROTEIN STRUCTURE
PROTEINS
PYRROLES
SACCHAROMYCES
SACCHAROMYCES CEREVISIAE
SCATTERING
TRYPTOPHAN
X-RAY DIFFRACTION
YEASTS