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 C -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 C -H bond in favor of C -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 C -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 C -H and C -H bonds of the substrate, and strongly strengthens the C -H bond in the substrate-bound orientation.