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

Abstract

The 2.2-.ANG. 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., and 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.fwdarw. Ile substitution in CCP(MI). A Trp-51 .fwdarw. 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., Fischel, L. A., English, A. M., Kraut, J., and 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 .fwdarw. 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., and 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 realtively large (ca. 1 .ANG.) 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.ANG.. 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. From the alteration of local structure that occurs in this mutant, coupled with the results of preliminary functional studies, we infer that Asp-235 exerts influence on the heme iron so as keep its sixth coordination site vacant, and hence reactive with peroxide substrate, over a wide pH range.