An Active Site Tyr Residue Guides the Regioselectivity of Lysine Hydroxylation by Nonheme Iron Lysine-4-hydroxylase Enzymes through Proton-Coupled Electron Transfer.

Lysine dioxygenase (KDO) is an important enzyme in human physiology involved in bioprocesses that trigger collagen cross-linking and blood pressure control. There are several KDOs in nature; however, little is known about the factors that govern the regio- and stereoselectivity of these enzymes. To understand how KDOs can selectively hydroxylate their substrate, we did a comprehensive computational study into the mechanisms and features of 4-lysine dioxygenase. In particular, we selected a snapshot from the MD simulation on KDO5 and created large QM cluster models (, , and ) containing 297, 312, and 407 atoms, respectively. The largest model predicts regioselectivity that matches experimental observation with rate-determining hydrogen atom abstraction from the C-H position, followed by fast OH rebound to form 4-hydroxylysine products. The calculations show that in model , the dipole moment is positioned along the C-H bond of the substrate and, therefore, the electrostatic and electric field perturbations of the protein assist the enzyme in creating C-H hydroxylation selectivity. Furthermore, an active site Tyr residue is identified that reacts through proton-coupled electron transfer akin to the axial Trp residue in cytochrome peroxidase. Thus, upon formation of the iron(IV)-oxo species in the catalytic cycle, the Tyr phenol loses a proton to the nearby Asp residue, while at the same time, an electron is transferred to the iron to create an iron(III)-oxo active species. This charged tyrosyl residue directs the dipole moment along the C-H bond of the substrate and guides the selectivity to the C-hydroxylation of the substrate.