Design of Heme Enzymes with a Tunable Substrate Binding Pocket Adjacent to an Open Metal Coordination Site

The catalytic versatility of pentacoordinated iron is
highlighted
by the broad range of natural and engineered activities of heme enzymes
such as cytochrome P450s, which position a porphyrin cofactor coordinating
a central iron atom below an open substrate binding pocket. This catalytic
prowess has inspired efforts to design de novo helical bundle scaffolds
that bind porphyrin cofactors. However, such designs lack the large
open substrate binding pocket of P450s, and hence, the range of chemical
transformations accessible is limited. Here, with the goal of combining
the advantages of the P450 catalytic site geometry with the almost
unlimited customizability of de novo protein design, we design a high-affinity
heme-binding protein, dnHEM1, with an axial histidine ligand, a vacant
coordination site for generating reactive intermediates, and a tunable
distal pocket for substrate binding. A 1.6 Å X-ray crystal structure
of dnHEM1 reveals excellent agreement to the design model with key
features programmed as intended. The incorporation of distal pocket
substitutions converted dnHEM1 into a proficient peroxidase with a
stable neutral ferryl intermediate. In parallel, dnHEM1 was redesigned
to generate enantiocomplementary carbene transferases for styrene
cyclopropanation (up to 93% isolated yield, 5000 turnovers, 97:3 e.r.)
by reconfiguring the distal pocket to accommodate calculated transition
state models. Our approach now enables the custom design of enzymes
containing cofactors adjacent to binding pockets with an almost unlimited
variety of shapes and functionalities.