1CCP

X-RAY STRUCTURES OF RECOMBINANT YEAST CYTOCHROME C PEROXIDASE AND THREE HEME-CLEFT MUTANTS PREPARED BY SITE-DIRECTED MUTAGENESIS

Summary for 1CCP

• Entry DOI: 10.2210/pdb1ccp/pdb
• Related: 1BEJ 1BEM 1BEQ 1BES 1CPD 1CPE 1CPF 1CPG 2CCP 3CCP 4CCP 5CCP 6CCP 7CCP
• Descriptor: YEAST CYTOCHROME C PEROXIDASE, PROTOPORPHYRIN IX CONTAINING FE (3 entities in total)
• Functional Keywords: oxidoreductase (h2o2 (a)), oxidoreductase
• Biological source: Saccharomyces cerevisiae (baker's yeast)
• Cellular location: Mitochondrion matrix: P00431
• Total number of polymer chains: 1
• Total formula weight: 34386.09

Authors

Wang, J.,Mauro, J.M.,Edwards, S.L.,Oatley, S.J.,Fishel, L.A.,Ashford, V.A.,Xuong, N.-H.,Kraut, J. (deposition date: 1990-02-28, release date: 1991-07-15, Last modification date: 2024-02-07)

Primary citation

Wang, J.M.,Mauro, M.,Edwards, S.L.,Oatley, S.J.,Fishel, L.A.,Ashford, V.A.,Xuong, N.H.,Kraut, J.
X-ray structures of recombinant yeast cytochrome c peroxidase and three heme-cleft mutants prepared by site-directed mutagenesis.
Biochemistry, 29:7160-7173, 1990

PubMed Abstract: 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)

PubMed: 2169873
DOI: 10.1021/bi00483a003

Experimental method

X-RAY DIFFRACTION (2.2 Å)