PDBsum entry 1aev

Oxidoreductase

PDB id: 1aev

Contents

Protein chain

291 a.a.

Ligands

HEM

AMT

Waters ×51

References listed in PDB file

Key reference

• Title: Introduction of novel substrate oxidation into cytochrome c peroxidase by cavity complementation: oxidation of 2-Aminothiazole and covalent modification of the enzyme.
• Authors: R.A.Musah, D.B.Goodin.
• Ref.: Biochemistry, 1997, 36, 11665-11674. [DOI no: 10.1021/bi9708038]
• PubMed id: 9305956

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.

Secondary reference #1

• Title: Small molecule binding to an artificially created cavity at the active site of cytochrome c peroxidase.
• Authors: M.M.Fitzgerald, M.J.Churchill, D.E.Mcree, D.B.Goodin.
• Ref.: Biochemistry, 1994, 33, 3807-3818. [DOI no: 10.1021/bi00179a004]
• PubMed id: 8142383

Secondary reference #2

• Title: The asp-His-Fe triad of cytochrome c peroxidase controls the reduction potential, Electronic structure, And coupling of the tryptophan free radical to the heme.
• Authors: D.B.Goodin, D.E.Mcree.
• Ref.: Biochemistry, 1993, 32, 3313-3324. [DOI no: 10.1021/bi00064a014]
• PubMed id: 8384877