Abstract
The chemical identification of the modified heme (the green heme) during chloroperoxidase catalyzed epoxidation of allylbenzene remains unestablished due to its high instability within the protein matrix, the absence of paramagnetically shifted signals, and the difficulty in obtaining crystals of the modified enzyme. We have established the unambiguous structure of the modified prosthetic heme group, which was extracted from the protein matrix using (2)D NMR spectroscopy and LC-MS spectrometry. The modified heme was isolated as a µ-oxo dimer that can be quantitatively converted to the corresponding monomer. The depolymerized green heme displayed characteristic NMR signatures of iron porphyrin complexes, but no Nuclear Overhauser Effect was observable to assist in signal assignment. An alternative strategy was applied by removing the iron center of the green heme, resulting in a stable demetallated green porphyrin species. Complete assignment of all the NMR resonances in the demetallated green heme allowed us to establish the molecular architecture of the modified species as a novel N-alkylated heme. Decisive space correlations between the propyl protons of allylbenzene and the γ meso proton coupled with clear dipolar connectivities between the propyl-2H of the substrate and the β proton in the side chain of the propionic acid at carbon-6 of the porphyrin ring, clearly indicate that allylbenzene was covalently attached to the nitrogen atom of the pyrrole ring III of the prosthetic heme. In this study, the mechanism of green CPO formation and its relation to CPO catalyzed chiral transformations are also discussed. It is concluded that the double-phenyl clamp formed by two phenylalanine residues at the distal heme pocket plays a critical role in fine-tuning substrate orientation that determines the outcome of CPO catalyzed epoxidation of substituted styrenes.