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 OsO4 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 FeCl2 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 Trp191 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 FeIII/FeII 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.
Original language | English (US) |
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Pages (from-to) | 635-643 |
Number of pages | 9 |
Journal | Journal of Inorganic Biochemistry |
Volume | 91 |
Issue number | 4 |
DOIs | |
State | Published - Sep 20 2002 |
Externally published | Yes |
Keywords
- Cytochrome c peroxidase
- Heme cd model
- Reconstituted oxochlorin
ASJC Scopus subject areas
- Biochemistry
- Inorganic Chemistry