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Mesopone cytochromecperoxidase: functional model of heme oxygenated oxidases

Authors
Journal
Journal of Inorganic Biochemistry
0162-0134
Publisher
Elsevier
Publication Date
Volume
91
Issue
4
Identifiers
DOI: 10.1016/s0162-0134(02)00447-6
Keywords
  • Hemecd1Model
  • Reconstituted Oxochlorin
  • Cytochromecperoxidase
Disciplines
  • Biology

Abstract

Abstract The effect of heme ring oxygenation on enzyme structure and function has been examined in a reconstituted cytochrome c peroxidase. Oxochlorin derivatives were formed by OsO 4 treatment of mesoporphyrin followed by acid-catalyzed pinacol rearrangement. The northern oxochlorin isomers were isolated by chromatography, and the regio-isomers assignments determined by 2D COSY and NOE 1H NMR. The major isomer, 4-mesoporphyrinone (Mp), was metallated with FeCl 2 and reconstituted into cytochrome c peroxidase (CcP) forming a hybrid green protein, MpCcP. The heme-altered enzyme has 99% wild-type peroxidase activity with cytochrome c. EPR spectroscopy of MpCcP intermediate compound I verifies the formation of the Trp 191 radical similar to wild-type CcP in the reaction cycle. Peroxidase activity with small molecules is varied: guaiacol turnover increases approximately five-fold while that with ferrocyanide is ∼85% of native. The electron-withdrawing oxo-substitutents on the cofactor cause a ∼60-mV increase in Fe III/Fe II reduction potential. The present investigation represents the first structural characterization of an oxochlorin protein with X-ray intensity data collected to 1.70 Å. Although a mixture of R- and S-mesopone isomers of the FeMP cofactor was used during heme incorporation into the apo-protein, only the S-isomer is found in the crystallized protein.

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