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 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.

Author Keywords: Heme cd₁ model; Reconstituted oxochlorin; Cytochrome c peroxidase

Article Outline

1. Introduction

2. Experimental procedures

2.1. Synthesis of dihydroxymesochlorin dimethyl ester (mesochlorindiol)

2.2. Synthesis of 4-mesoporphyrinone dimethyl ester (mesopone or 8,12,-diethyl-3,8,13,17-tetramethyl-7-oxo-prophyrin-2,18-diproprionic acid dimethyl ester)

2.3. Synthesis of Fe^III-Cl mesopone

2.4. Protein expression, reconstitution and purification

2.5. Crystallization, X-ray data collection and structure refinement

2.6. Steady state activity assays

2.7. Voltammetry

2.8. Electron paramagnetic resonance (EPR) spectroscopy

3. Results and discussion

4. Conclusion

5. Abbreviations

Acknowledgements

References