Non-Electrostatic Basis for an Artificial Metalloenzyme Catalysis

The artificial metalloenzyme (referred to as Ir-Q ) reported by Hartwig and coworkers presented an important milestone in merging the extraordinary efficiency of biocatalyst with the versatility of small molecule chemical catalyst in catalyzing a new-to-nature carbene insertion reaction. The artificial enzyme results from formal replacement of the Fe by an Ir(Me) moiety along with four C317G, T213G, L69V, V254L mutations in a natural Cytochrome enzyme CYP119 by directed evolution method. Importantly, this is a show-stopper enzyme as it exhibits a catalytic rate enhancement similar to that of natural enzymes. Despite this remarkable discovery, there is no mechanistic understanding as to why it displays extraordinary efficiency, so far been intractable to experimental methods. In this study, we have deciphered the ‘catalytically active conformation’ of Ir-Q using large-scale molecular dynamics simulations and rigorous quantum chemical calculations. Our study reveals how directed evolution mutations precisely position the cofactor-substrate in an unusual orientation within a reshaped active site that emerged during evolution and fostered by C−H…π interactions from more ordered mutated L69V and V254L residues. This productive conformation correctly reproduces the experimental barrier height and the catalytic effect of 2.7 kcal/mol, in excellent agreement with observed rate enhancement. Moreover, the active conformation features an unprecedented bonding interaction in a metal-carbene species that preferentially stabilizes the rate determining formation of an Iridium-Porphyrin Carbene intermediate to render the observed high catalytic rate acceleration. While the electrostatic criteria are widely established, this study suggests a new design paradigm towards realization of fully programmable protein catalysis.