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

Abstract

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. File list (2) download file view on ChemRxiv MnDPP_manuscript_092820_FINAL_chemrxiv.pdf (6.62 MiB) download file view on ChemRxiv MnDPP_SI_092820_FINAL.pdf (4.08 MiB) De novo design, solution characterization, and crystallographic structure of an abiological Mn-porphyrin binding protein capable of stabilizing a Mn(V) species

Rational design of photosynthetic reaction center protein maquettes

“…Antiparallel four-helix bundles that bind porphyrins are common in nature ( Hunte et al, 2000 ; Malone et al, 2019 ; Mathews et al, 1979 ; Weber et al, 1980 ; Yankovskaya et al, 2003 ; Dautant et al, 1998 ), and experimental structures of de novo -designed versions have been published recently ( Polizzi et al, 2017 ; Pirro et al, 2020 ; Mann et al, 2021 ; Hutchins et al, 2020 ). One common trend in all of the examples cited here is that the plane of the porphyrin ring cuts through the e - e interface of the helical bundle, causing the e - e interface to expand while the g - g interface can remain narrow.…”

Artificial Metalloproteins: At the Interface between Biology and Chemistry

“…In one recent report, he was successful in installing a synthetic Mn–porphyrin in a de novo designed four-helix bundle protein that also incorporated an access channel for external species that were necessary for catalytic function. 93 The starting point in the development was a four-helix bundle protein (PS1) that previously had been used to bind a redox-inactive Zn–porphyrin cofactor. 94 Computational studies on PS1 led to the new protein MPP1 (manganese porphyrin-binding protein 1), which could accommodate a larger [Mn(diphenylporphyrin)] n (Mn(dpp)) cofactor.…”

Mechanistic Studies on the Epoxidation of Alkenes by Macrocyclic Manganese Porphyrin Catalysts

“…[3] The enzyme contains a binding pocket, which facilitates the formation of the oxospecies and increases the catalytic efficiency of the reaction. [4][5][6] Such a cavity-driven catalysis [7,8] is an important aspect that should be considered if one wants to design and construct synthetic systems mimicking the action of cytochrome P450 enzymes. [9] A possible mimic of a binding pocket with a nearby catalytic reaction site is the porphyrin cage compound H 2 C (Figure 1).…”

“…The binding of a substrate initiates a series of chemical events, in which molecular oxygen is activated by the heme to generate a high valent iron‐oxo complex, which transfers its oxygen to the bound substrate [3] . The enzyme contains a binding pocket, which facilitates the formation of the oxo‐species and increases the catalytic efficiency of the reaction [4–6] . Such a cavity‐driven catalysis [7,8] is an important aspect that should be considered if one wants to design and construct synthetic systems mimicking the action of cytochrome P450 enzymes [9] …”