Borovik, A.S.; Hendrich, M.P.; Moënne-Loccoz, P.

J. Am. Chem. Soc. 2021, 143, 2384-2393, 10.1021/jacs.0c12564

Dinuclear iron centers with a bridging hydroxido or oxido ligand form active sites within a variety of metalloproteins. A key feature of these sites is the ability of the protein to control the structures around the Fe centers, which leads to entatic states that are essential for function. To simulate this controlled environment, artificial proteins have been engineered using biotin–streptavidin (Sav) technology in which Fe complexes from adjacent subunits can assemble to form [FeIII–(μ-OH)–FeIII] cores. The assembly process is promoted by the site-specific localization of the Fe complexes within a subunit through the designed mutation of a tyrosinate side chain to coordinate the Fe centers. An important outcome is that the Sav host can regulate the Fe···Fe separation, which is known to be important for function in natural metalloproteins. Spectroscopic and structural studies from X-ray diffraction methods revealed uncommonly long Fe···Fe separations that change by less than 0.3 Å upon the binding of additional bridging ligands. The structural constraints imposed by the protein host on the di-Fe cores are unique and create examples of active sites having entatic states within engineered artificial metalloproteins.

Brustad, E.M.; Nicewicz, D.A.

ChemBioChem 2020, 21, 3146-3150, 10.1002/cbic.202000362

A pair of 9-mesityl-10-phenyl acridinium (Mes−Acr+) photoredox catalysts were synthesized with an iodoacetamide handle for cysteine bioconjugation. Covalently tethering of the synthetic Mes−Acr+ cofactors with a small panel of thermostable protein scaffolds resulted in 12 new artificial enzymes. The unique chemical and structural environment of the protein hosts had a measurable effect on the photophysical properties and photocatalytic activity of the cofactors. The constructed Mes−Acr+ hybrid enzymes were found to be active photoinduced electron-transfer catalysts, controllably oxidizing a variety of aryl sulfides when irradiated with visible light, and possessed activities that correlated with the photophysical characterization data. Their catalytic performance was found to depend on multiple factors including the Mes−Acr+ cofactor, the protein scaffold, the location of cofactor immobilization, and the substrate. This work provides a framework toward adapting synthetic photoredox catalysts into artificial cofactors and includes important considerations for future bioengineering efforts.

Sauer, D.F.; Schwaneberg, U.

Nat. Catal. 2021, 4, 814-827, 10.1038/s41929-021-00673-3

The field of artificial metalloenzymes (ArMs) is rapidly growing and ArMs are attracting increasing attention, for example, in the fields of biosensing and drug therapy. Protein-engineering methods that are commonly used to tailor the properties of natural enzymes are more frequently included in the design of ArMs. In particular, directed evolution allows the fine-tuning of ArMs, ultimately assisting in the development of their enormous potential. The integration of ArMs in whole cells enables their in vivo application and facilitates high-throughput directed-evolution methodologies. In this Review, we highlight the recent progress of whole-cell conversions and applications of ArMs and critically discuss their limitations and prospects. To focus on ArMs and their specific properties, advantages and challenges, the evolution of natural enzymes for non-natural reactions will not be covered.

Okuda, J.; Sauer, D.F.

Beilstein J. Org. Chem. 2018, 14, 2861-2871, 10.3762/bjoc.14.265

This review summarizes the recent progress of Grubbs–Hoveyda (GH) type olefin metathesis catalysts incorporated into the robust fold of β-barrel proteins. Anchoring strategies are discussed and challenges and opportunities in this emerging field are shown from simple small-molecule transformations over ring-opening metathesis polymerizations to in vivo olefin metathesis.

Brustad, E.M.

ChemBioChem 2017, 18, 2380-2384, 10.1002/cbic.201700397

Engineering an (Ir)regular cytochrome P450: Mutations within the heme‐binding pocket of a cytochrome P450 enabled the selective incorporation of an artificial Ir‐porphyrin cofactor into the protein, in cells. This orthogonal metalloprotein showed enhanced behavior in unnatural carbene‐mediated cyclopropanation of aliphatic and electron‐deficient olefins.