Electrostatic Perturbations from the Protein Affect C−H Bond Strengths of the Substrate and Enable Negative Catalysis in the TmpA Biosynthesis Enzyme

The nonheme iron dioxygenase 2‐(trimethylammonio)‐ethylphosphonate dioxygenase (TmpA) is an enzyme involved in the regio‐ and chemoselective hydroxylation at the C1‐position of the substrate as part of the biosynthesis of glycine betaine in bacteria and carnitine in humans. To understand how the enzyme avoids breaking the weak C2−H bond in favor of C1‐hydroxylation, we set up a cluster model of 242 atoms representing the first and second coordination sphere of the metal center and substrate binding pocket, and investigated possible reaction mechanisms of substrate activation by an iron(IV)‐oxo species by density functional theory methods. In agreement with experimental product distributions, the calculations predict a favorable C1‐hydroxylation pathway. The calculations show that the selectivity is guided through electrostatic perturbations inside the protein from charged residues, external electric fields and electric dipole moments. In particular, charged residues influence and perturb the homolytic bond strength of the C1−H and C2−H bonds of the substrate, and strongly strengthens the C2−H bond in the substrate‐bound orientation. Density functional theory studies on large active site cluster models of the nonheme iron dioxygenase TmpA elucidated the origin of its chemoselectivity and identify it as negative catalysis, where an otherwise favorable channel is blocked. The enzyme succeeds in this thanks to charged residues in the substrate binding pocket, which produce a local electric dipole moment (μD) and electric field and these electrostatic perturbations guide the reaction to a selective reaction mechanism.