Introduction of Novel Substrate Oxidation into Cytochrome c Peroxidase by Cavity Complementation: Oxidation of 2-Aminothiazole and Covalent Modification of the Enzyme

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• 作者：Rabi A. Musah and David B. Goodin
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：September 30, 1997
• 年：1997
• 卷：36
• 期：39
• 页码：11665 - 11674
• 全文大小：303K
• 年卷期：v.36,no.39(September 30, 1997)
• ISSN：1520-4995

文摘

The binding and oxidation of an artificial substrate,2-aminothiazole, by an engineered cavityof cytochrome c peroxidase is described. The W191Gmutant has been shown to create a buried cavityinto which a number of small heterocyclic compounds will bind[Fitzgerald, M. M., Churchill, M. J.,McRee, D. E., & Goodin, D. B. (1994) Biochemistry33, 3807-3818], providing a specific site neartheheme from which substrates might be oxidized. In this study, weshow by titration calorimetry that2-aminothiazole binds to W191G with a K_d of0.028 mM at pH 6. A crystal structure at 2.3 Åresolutionof W191G in the presence of 2-aminothiazole reveals the occupation ofthis compound in the cavity, andindicates that it is in van der Waals contact with the heme. TheWT enzyme reacts with H₂O₂ to formCompound ES, in which both the iron center and the Trp-191 side chainare reversibly oxidized. For theW191F (and perhaps the W191G) mutants, the iron is still oxidized, butthe second equivalent existstransiently as a radical on the porphyrin before migrating to analternate protein radical site [Erman, J. E.,Vitello, L. B., Mauro, J. M., & Kraut, J. (1989)Biochemistry 28, 7992-7995]. Two separatereactionsare observed between 2-aminothiazole and the oxidized centers of W191G.In the one reaction, opticaland EPR spectra of the heme are used to show that 2-aminothiazole actsas an electron donor to the ferryl(Fe⁴⁺=O) center of W191G to reduce it to the ferricoxidation state. This reaction occurs from withinthe cavity, as it is not observed for variants that lack thisartificial binding site. A second reaction between2-aminothiazole and peroxide-oxidized W191G, which is much lessefficient, results in the specific covalentmodification of Tyr-236. Electrospray mass spectra of the W191Gafter incubation in 2-aminothiazoleand H₂O₂ show a modification of the proteinindicative of covalent binding of 2-aminothiazole. Thesiteof modification was determined to be Tyr-236 by CNBr peptide mappingand automated peptide sequencing.The covalent modification is only observed for W191G and W191Fwhich form the alternate radicalcenter. This observation provides an unanticipated assignment ofthis free radical species to Tyr-236,which is consistent with previous proposals that it is a tyrosine.The oxidation of 2-aminothiazole byW191G represents an example of how the oxidative capacity inherent inthe heme prosthetic group andthe specific binding behavior of artificial protein cavities can beharnessed and redirected toward theoxidation of organic substrates.