Halogen-Bonding-like Interactions Complement Hydrogen Bonding in a Human Flavin-Dependent Dehalogenase

An accurate description of halogen bonding in biomolecules remains a challenge in modern biochemistry, as exemplified by the flavin-dependent human dehalogenase, iodotyrosine deiodinase. Analyses of the static crystal structure and kinetics data suggested that halogen bonding is absent in the enzyme's active site, which does not preclude its transient formation along the binding or unbinding pathway. No studies have yet explored iodotyrosine deiodinase's dynamic behavior from the perspective of halogen bonding. Here, extensive equilibrium and nonequilibrium molecular dynamics (MD) simulations together with quantum-mechanical calculations are performed to elucidate how the cooperative interplay of transient halogen bonds and stable hydrogen bonds governs substrate recognition and retention. An extra-point charge model is employed to describe σ-hole of the halogen to capture its charge anisotropy in classical simulation. The isotropic steered molecular dynamics simulations and dynamic time warping algorithm identify dominant unbinding pathways for I-Tyr, Cl-Tyr, and I-Phenol. Substrate release kinetics is controlled by a stepwise disruption of key hydrogen bonds and salt bridges with K182, Y161, E157, and the flavin cofactor. Molecular dynamics simulation-derived geometries were screened, representative poses were chosen based on structural criteria, and halogen-bond-like interactions were validated through natural bond orbital and energy-decomposition analyses. Geometries consistent with halogen-bond-like interactions (0.5-2.0 kcal/mol) are observed for I-Tyr and I-Phenol near the protein surface. In contrast, Cl-Tyr interactions identified solely by structural criteria largely correspond to false positives, underscoring the necessity of quantum-mechanical validation for reliably characterizing halogen-bond-like interactions. These early, short-lived halogen bonds help orient the native substrate toward a possible alternate site, triggering a communication network that grows with strong hydrogen bonds during substrate recognition. By integrating dynamic simulations with quantum-mechanical validation, this work reconciles conflicting views on halogen bonding in human iodotyrosine deiodinase, revealing transient geometries consistent with halogen-bond-like interactions alongside persistent hydrogen bonds.