Mechanistic Manifold in a Hemoprotein-Catalyzed Cyclopropanation Reaction with Diazoketone

Abstract

Abstract Hemoproteins have recently emerged as a promising class of biological catalysts for promoting carbene transfer reactions not found in nature. Despite this progress, our mechanistic understanding of the interplay between productive and unproductive pathways in these reactions is limited. Using a combination of spectroscopic, structural, and computational methods, we have investigated the mechanism of a myoglobin-catalyzed cyclopropanation reaction with diazoketones. Our studies shed light into the nature and kinetics of key catalytic steps in this reaction, including formation of an early heme-bound diazo complex intermediate, the rate-determining nature of carbene formation, and the mechanism of the cyclopropanation step. Importantly, our studies reveal the existence of a complex mechanistic manifold behind this hemoprotein-catalyzed cyclopropanation, wherein the cyclopropanation pathway competes with alternative pathways, including formation of an N-bound carbene adduct of the protein heme cofactor, which was isolated and characterized by X-ray crystallography, UV-Vis, and Mössbauer spectroscopy. This species is able to regenerate the active biocatalyst, thus constituting a non-productive, yet non-destructive detour from the main catalytic cycle. These findings improve our understanding of biocatalytic cyclopropanations and the ensuing mechanistic picture is expected to offer a blueprint for both the mechanistic analysis of other hemoprotein-catalyzed carbene transfer reactions and the design and engineering of carbene transferases.