title: Mesopone cytochrome c peroxidase: functional model of heme oxygenated oxidases.
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 approximately 85% of native. The electron-withdrawing oxo-substitutents on the cofactor cause a approximately 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 A. 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.
authors.affiliation: Department of Chemistry, University of California, Irvine, CA 92697-2025, USA.
authors.forename: Chad E
authors.initials: CE
authors.lastname: Immoos
authors.forename: B
authors.initials: B
authors.lastname: Bhaskar
authors.forename: Michael S
authors.initials: MS
authors.lastname: Cohen
authors.forename: Tiffany P
authors.initials: TP
authors.lastname: Barrows
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Farmer
authors.forename: T L
authors.initials: TL
authors.lastname: Poulos
mesh_terms: D003001:Cloning, Molecular; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000378:metabolism*; D004563:Electrochemistry / Q000379:methods; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014961:X-Ray Diffraction / Q000379:methods
chemical_list: D011994:Recombinant Proteins; D006418:Heme; D003578:Cytochrome-c Peroxidase