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

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)

Enzyme-catalyzed Mechanism of Isoniazid Activation in Class I and Class III Peroxidases

“…The acyl radical may diffuse from the active site or may remain oriented near the ␦-meso edge of the heme. (2), orange for the APX crystal structure (29), and light green for the CcP crystal structure (30). The conserved water molecule on the distal side of the active site above the heme is shown in red (w1354 for HRPC, w501 for APX, and w595 for CcP).…”

“…Furthermore, we have utilized information about the HRPC⅐INH complex in combination with crystal structures of mtCP (2) and the class I peroxidases APX (13,29) and CcP (30) to predict the binding mode of INH in these enzymes, which have all been shown to turnover the drug. Using available mechanistic data about the activation pathway involving the formation of the isonicotinamide end product (8), we propose an enzyme-catalyzed mechanism for INH activation that can yield the acyl radical thought to be the activated form of the drug.…”

“…Computational Predictions of INH Binding Sites-GRID calculations (52) were performed on atomic coordinate files of the crystal structures of APX (53) and CcP (30). INH was docked into the active site of the crystal structures of rsAPX (29) and CcP (30) based upon a superposition of each of these crystal structures with the HRPC⅐INH NMRderived model using all atoms of the heme moiety, the conserved distal histidine and arginine residues, and the conserved proximal histidine and aspartic acid residues.…”

“…In addition, we report data demonstrating that other class I enzymes, ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), are also capable of turning over INH. Furthermore, the availability of an NMR-derived HRPC⅐INH complex enables us to generate docked complexes of INH in the active sites of the crystal structures of APX (29) and CcP (30) in the same manner as described for the mtCP⅐INH complex (2). Using these complexes potential enzyme-drug interactions found in the class I and class III peroxidases are described, and an enzyme-catalyzed mechanism for INH activation is proposed.…”

The role of aspartate-235 in the binding of cations to an artificial cavity at the radical site of cytochromecperoxidase

“…Of the mutants of Asp-235 that have been described, only D235E, which retains a carboxylate side chain, has been shown to form a stable Trp-191 radical (Fishel et al, 1991;Goodin & McRee, 1993). However, because the tryptophan ring conformation is significantly altered in the D235N and D235A mutants (Wang et al, 1990;Goodin & McRee, 1993), the direct role of Asp-235 in the stabilization of the Trp-191 radical has not been clearly defined.…”