De Novo Design, Solution Characterization, and Crystallographic Structure of an Abiological Mn-Porphyrin Binding Protein Capable of Stabilizing a Mn(V) Species

De novo protein design offers the opportunity to test our understanding of how metalloproteins perform difficult transformations. Attaining high-resolution structural information is critical to understanding how such designs function. There have been many successes in the design of porphyrin-binding proteins, however crystallographic characterization has been elusive, limiting what can be learned from such studies as well as the extension to new functions. Moreover, formation of highly oxidizing high-valent intermediates poses design challenges that have not been previously implemented: 1) purposeful design of substrate/oxidant access to the binding site and 2) limiting deleterious oxidation of the protein scaffold. Here we report the first crystallographically characterized porphyrin-binding protein that was programmed to not only bind a synthetic Mn-porphyrin but also maintain binding site access to form high-valent oxidation states. We explicitly designed a binding site with accessibility to dioxygen units in the open coordination site of the Mn center. In solution, the protein is capable of accessing a high-valent Mn(V)-oxo species which can transfer an O-atom to a thioether substrate. The crystallographic structure is within 0.6 Å of the design, and indeed contained an aquo ligand with a second water molecule stabilized by hydrogen-bonding to a Gln sidechain in the active site, offering a structural explanation for the observed reactivity.