1ccp
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X-RAY STRUCTURES OF RECOMBINANT YEAST CYTOCHROME C PEROXIDASE AND THREE HEME-CLEFT MUTANTS PREPARED BY SITE-DIRECTED MUTAGENESIS
OverviewOverview
The 2.2-A X-ray structure for CCP(MI), a plasmid-encoded form of, Saccharomyces cerevisiae cytochrome c peroxidase (CCP) expressed in, Escherichia coli [Fishel, L.A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360], 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----Ile substitution, in CCP(MI). A Trp-51----Phe mutant remains pentacoordinated and exhibits, only minor distal structural adjustments. 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 [Smulevich, G., Mauro, J.M., Fishel, L. A., English, A. M., Kraut, J., & Spiro, T. G. (1988) Biochemistry 27, 5477-5485]. 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----Phe mutant that has substantially diminished enzyme activity, and altered magnetic properties [Mauro, J. M., Fishel, L. F., Hazzard, J., T., Meyer, T. E., Tollin, G., Cusanovich, M. A., & Kraut, J. (1988), Biochemistry 27, 6243-6256] 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 (ca. 1 A), 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 structure, rules out a major alteration in protein conformation as a reason for the, dramatically decreased activity of the W191F mutant. Changing proximal, Asp-235 to Asn results in two significant localized structural changes., First, the heme iron moves toward the porphyrin plane, and distal water, 595 now clearly resides in the iron coordination sphere at a distance of, 2.0 A. The observation of hexacoordinated iron for the D235N mutant is in, accord with previous resonance Raman results. Second, the indole side, chain of Trp-191 has flipped over as a result of the mutation; the, tryptophan N epsilon takes part in a new hydrogen bond with the backbone, carbonyl oxygen of Leu-177.(ABSTRACT TRUNCATED AT 400 WORDS)
About this StructureAbout this Structure
1CCP is a Single protein structure of sequence from Saccharomyces cerevisiae with HEM as ligand. Active as Cytochrome-c peroxidase, with EC number 1.11.1.5 Full crystallographic information is available from OCA.
ReferenceReference
X-ray structures of recombinant yeast cytochrome c peroxidase and three heme-cleft mutants prepared by site-directed mutagenesis., Wang JM, Mauro M, Edwards SL, Oatley SJ, Fishel LA, Ashford VA, Xuong NH, Kraut J, Biochemistry. 1990 Aug 7;29(31):7160-73. PMID:2169873
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