1aev
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INTRODUCTION OF NOVEL SUBSTRATE OXIDATION INTO CYTOCHROME C PEROXIDASE BY CAVITY COMPLEMENTATION: OXIDATION OF 2-AMINOTHIAZOLE AND COVALENT MODIFICATION OF THE ENZYME (2-AMINOTHIAZOLE)
OverviewOverview
The binding and oxidation of an artificial substrate, 2-aminothiazole, by, an engineered cavity of cytochrome c peroxidase is described. The W191G, mutant has been shown to create a buried cavity into which a number of, small heterocyclic compounds will bind [Fitzgerald, M. M., Churchill, M., J., McRee, D. E., & Goodin, D. B. (1994) Biochemistry 33, 3807-3818], providing a specific site near the heme from which substrates might be, oxidized. In this study, we show by titration calorimetry that, 2-aminothiazole binds to W191G with a Kd of 0.028 mM at pH 6. A crystal, structure at 2.3 A resolution of W191G in the presence of 2-aminothiazole, reveals the occupation of this compound in the cavity, and indicates that, it is in van der Waals contact with the heme. The WT enzyme reacts with, H2O2 to form Compound ES, in which both the iron center and the Trp-191, side chain are reversibly oxidized. For the W191F (and perhaps the W191G), mutants, the iron is still oxidized, but the second equivalent exists, transiently as a radical on the porphyrin before migrating to an alternate, protein radical site [Erman, J. E., Vitello, L. B., Mauro, J. M., & Kraut, J. (1989) Biochemistry 28, 7992-7995]. Two separate reactions are observed, between 2-aminothiazole and the oxidized centers of W191G. In the one, reaction, optical and EPR spectra of the heme are used to show that, 2-aminothiazole acts as an electron donor to the ferryl (Fe4+&dbd;O), center of W191G to reduce it to the ferric oxidation state. This reaction, occurs from within the cavity, as it is not observed for variants that, lack this artificial binding site. A second reaction between, 2-aminothiazole and peroxide-oxidized W191G, which is much less efficient, results in the specific covalent modification of Tyr-236. Electrospray, mass spectra of the W191G after incubation in 2-aminothiazole and H2O2, show a modification of the protein indicative of covalent binding of, 2-aminothiazole. The site of modification was determined to be Tyr-236 by, CNBr peptide mapping and automated peptide sequencing. The covalent, modification is only observed for W191G and W191F which form the alternate, radical center. This observation provides an unanticipated assignment of, this free radical species to Tyr-236, which is consistent with previous, proposals that it is a tyrosine. The oxidation of 2-aminothiazole by W191G, represents an example of how the oxidative capacity inherent in the heme, prosthetic group and the specific binding behavior of artificial protein, cavities can be harnessed and redirected toward the oxidation of organic, substrates.
About this StructureAbout this Structure
1AEV is a Single protein structure of sequence from Saccharomyces cerevisiae with HEM and AMT as ligands. Active as Cytochrome-c peroxidase, with EC number 1.11.1.5 Structure known Active Site: AVE. Full crystallographic information is available from OCA.
ReferenceReference
Introduction of novel substrate oxidation into cytochrome c peroxidase by cavity complementation: oxidation of 2-aminothiazole and covalent modification of the enzyme., Musah RA, Goodin DB, Biochemistry. 1997 Sep 30;36(39):11665-74. PMID:9305956
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