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

Abstract

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 (A, B, and C) containing 297, 312, and 407 atoms, respectively. The largest model predicts regioselectivity that matches experimental observation with rate-determining hydrogen atom abstraction from the C₄-H position, followed by fast OH rebound to form 4-hydroxylysine products. The calculations show that in model C, the dipole moment is positioned along the C₄-H bond of the substrate and, therefore, the electrostatic and electric field perturbations of the protein assist the enzyme in creating C₄-H hydroxylation selectivity. Furthermore, an active site Tyr₂₃₃ residue is identified that reacts through proton-coupled electron transfer akin to the axial Trp residue in cytochrome c peroxidase. Thus, upon formation of the iron(IV)-oxo species in the catalytic cycle, the Tyr₂₃₃ phenol loses a proton to the nearby Asp₁₇₉ 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 C₄-H bond of the substrate and guides the selectivity to the C₄-hydroxylation of the substrate.

Figure 1

Active site of KDO5 (PDB ID: 6EUR) and the general reaction catalyzed by the enzyme.

Figure 2

Cluster models A, B, and C of KDO5 as studied in this work. Model C is based on model B with the second-coordination sphere expanded, as highlighted in purple. Wiggly lines identify where bonds were cut and replaced by C–H.

Figure 3

(a) RMSD plot of the MD simulation on a substrate-bound KDO5 structure. (b) Distance plots of key active site amino acid residues with respect to the substrate. (c) Substrate–oxidant C₃–O, C₄–O, and C₅–O distances during the MD simulation.

Figure 4

(a) UB3LYP-D3/BS1-optimized geometries of the iron(IV)-oxo species of KDO5 for models A, B, and C with bond lengths in Å. (b) Overlay of the ⁵Re_C structure (blue) with the crystal structure coordinates of the 6EUR PDB (gold).

Figure 5

UB3LYP/BS1//UB3LYP/BS2-calculated free energy profile for Lys hydroxylation at the C₄–H (red) and C₅–H (blue) positions in KDO5. Energies are in kcal mol^–1 with ΔE + ZPE (outside parentheses) and ΔG (in parentheses). Optimized transition state geometries give bond lengths in Å and angles in degrees.

Figure 6

Electronic configuration of the intermediates along the hydroxylation mechanism for model C.

Figure 7

Structure comparison of KDO5 (6EUR PDB file) with asparagine hydroxylase (2OG7 PDB).

Figure 8

(a) Electric dipole moment in ⁵Re_C. (b) Electric field effects along the x- or z-direction on the C–H BDEs of an isolated lysine substrate molecule with ΔG values (in kcal mol^–1) as a function of the applied electric field.