Mesopone cytochrome c peroxidase: functional model of heme oxygenated oxidases

摘要

The effect of heme ring oxygenation on enzyme structure and function has been examined in a reconstituted cytochrome c peroxidase. Oxochlorin defivatives 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 H-1 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 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 similar to85% of native. The electron-withdrawing oxo-substitutents on the cofactor cause a similar to60-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 Angstrom. 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. (C) 2002 Elsevier Science Inc. All rights reserved. [References: 41]