Electrostatic Perturbations from the Protein Affect C-H Bond Strengths of the Substrate and Enable Negative Catalysis...

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.