Activation and Hydroxylation Mechanism of Aromatic C-H and C-F of 3-Fluoro-l-tyrosine Catalyzed by the Heme-Dependent Tyrosine Hydroxylase.

Abstract

LmbB2 is a peroxygenase-like heme-dependent l-tyrosine hydroxylase (TyrH) that hydroxylates natural l-tyrosine to L-3,4-dihydroxyphenylalanine (DOPA). When challenged with 3-fluoro-l-tyrosine as a substrate, both the C-H and C-F bonds can be hydroxylated, leading to two products, DOPA and 3-F-5-OH-Tyr. However, the crystal structure shows only one binding conformation of the substrate (3-F-Tyr) but two orientations of the fluorine atom, which means that both C-H and C-F are activated. To gain insights into the hydroxylation mechanism, computational models were constructed, and a series of combined QM/MM calculations were performed. Our calculation results reveal that it is the two binding orientations of the substrate that control the final product distribution. Orientations A and B employ different mechanisms for breaking C-H and C-F as well as for hydroxylating the aromatic substrate. Orientation A only corresponds to the C-H hydroxylation, while orientation B is associated with the C-F hydroxylation. The dissociation of the O-O bond in Cpd 0 (Fe(III)-OOH) is in concert with the electron transfer from the iron center to the porphyrin ring, generating the Cpd I intermediate, which is responsible for initiating the reaction. Since the leaving F- takes two electrons away from the substrate, another molecule of hydrogen peroxide is required to complete the catalytic cycle in hydroxylation of C-F bond, and the aromatization of intermediate may occur outside the active site of the enzyme. During the reaction, His88, two crystal water molecules, and the porphyrin ring play critical roles in the proton and electron transfer. Although the hydroxylation of C-H and C-F bonds follows different reaction pathways, they correspond to very similar overall energy barriers; therefore, it is the distribution of two binding orientations of the substrate that determines the final hydroxylated products. These results may provide useful information for understanding the reactions catalyzed by heme-dependent tyrosine hydroxylases.