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Publication Abstract from PubMed

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 A of the design and indeed contained an aquo ligand with a second water molecule stabilized by hydrogen bonding to a Gln side chain in the active site, offering a structural explanation for the observed reactivity.

De Novo Design, Solution Characterization, and Crystallographic Structure of an Abiological Mn-Porphyrin-Binding Protein Capable of Stabilizing a Mn(V) Species.,Mann SI, Nayak A, Gassner GT, Therien MJ, DeGrado WF J Am Chem Soc. 2021 Jan 13;143(1):252-259. doi: 10.1021/jacs.0c10136. Epub 2020, Dec 29. PMID:33373215^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.