1AEV

INTRODUCTION OF NOVEL SUBSTRATE OXIDATION INTO CYTOCHROME C PEROXIDASE BY CAVITY COMPLEMENTATION: OXIDATION OF 2-AMINOTHIAZOLE AND COVALENT MODIFICATION OF THE ENZYME (2-AMINOTHIAZOLE)

1AEV の概要

• エントリーDOI: 10.2210/pdb1aev/pdb
• 分子名称: CYTOCHROME C PEROXIDASE, PROTOPORPHYRIN IX CONTAINING FE, 2-AMINOTHIAZOLE, ... (4 entities in total)
• 機能のキーワード: oxidoreductase, peroxidase, transit peptide
• 由来する生物種: Saccharomyces cerevisiae (baker's yeast)
• 細胞内の位置: Mitochondrion matrix: P00431
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 34175.87

構造登録者

Musah, R.A.,Fitzgerald, M.M.,Jensen, G.M.,Mcree, D.E.,Goodin, D.B. (登録日: 1997-02-25, 公開日: 1997-09-04, 最終更新日: 2024-05-22)

主引用文献

Musah, R.A.,Goodin, D.B.
Introduction of novel substrate oxidation into cytochrome c peroxidase by cavity complementation: oxidation of 2-aminothiazole and covalent modification of the enzyme.
Biochemistry, 36:11665-11674, 1997

PubMed Abstract: 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.

PubMed: 9305956
DOI: 10.1021/bi9708038

実験手法

X-RAY DIFFRACTION (2.1 Å)