Borovik, A.S.; Don Tilley, T.

J. Am. Chem. Soc. 2018, 140, 2739-2742, 10.1021/jacs.7b13052

Artificial metalloproteins (ArMs) containing Co4O4 cubane active sites were constructed via biotin–streptavidin technology. Stabilized by hydrogen bonds (H-bonds), terminal and cofacial CoIII–OH2 moieties are observed crystallographically in a series of immobilized cubane sites. Solution electrochemistry provided correlations of oxidation potential and pH. For variants containing Ser and Phe adjacent to the metallocofactor, 1e–/1H+ chemistry predominates until pH 8, above which the oxidation becomes pH-independent. Installation of Tyr proximal to the Co4O4 active site provided a single H-bond to one of a set of cofacial CoIII–OH2 groups. With this variant, multi-e–/multi-H+ chemistry is observed, along with a change in mechanism at pH 9.5 that is consistent with Tyr deprotonation. With structural similarities to both the oxygen-evolving complex of photosystem II (H-bonded Tyr) and to thin film water oxidation catalysts (Co4O4 core), these findings bridge synthetic and biological systems for water oxidation, highlighting the importance of secondary sphere interactions in mediating multi-e–/multi-H+ reactivity.

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.

Borovik, A.S.

J. Am. Chem. Soc. 2017, 139, 17289-17292, 10.1021/jacs.7b10452

Copper–hydroperoxido species (CuII–OOH) have been proposed to be key intermediates in biological and synthetic oxidations. Using biotin–streptavidin (Sav) technology, artificial copper proteins have been developed to stabilize a CuII–OOH complex in solution and in crystallo. Stability is achieved because the Sav host provides a local environment around the Cu–OOH that includes a network of hydrogen bonds to the hydroperoxido ligand. Systematic deletions of individual hydrogen bonds to the Cu–OOH complex were accomplished using different Sav variants and demonstrated that stability is achieved with a single hydrogen bond to the proximal O-atom of the hydroperoxido ligand: changing this interaction to only include the distal O-atom produced a reactive variant that oxidized an external substrate.

Matsuo, T.

Chem. - Asian J. 2011, 6, 2491-2499, 10.1002/asia.201100107

H64D myoglobin mutant was reconstituted with two different types of synthetic hemes that have aromatic rings and a carboxylate‐based cluster attached to the terminus of one or both of the heme‐propionate moieties, thereby forming a “single‐winged cofactor” and “double‐winged cofactor,” respectively. The reconstituted mutant myoglobins have smaller Km values with respect to 2‐methoxyphenol oxidation activity relative to the parent mutant with native heme. This suggests that the attached moiety functions as a substrate‐binding domain. However, the kcat value of the mutant myoglobin with the double‐winged cofactor is much lower than that of the mutant with the native heme. In contrast, the mutant reconstituted with the single‐winged cofactor has a larger kcat value, thereby resulting in overall catalytic activity that is essentially equivalent to that of the native horseradish peroxidase. Enhanced peroxygenase activity was also observed for the mutant myoglobin with the single‐winged cofactor, thus indicating that introduction of an artificial substrate‐binding domain at only one of the heme propionates in the H64D mutant is the optimal engineering strategy for improving the peroxidase activity of myoglobin.

Matsuo, T.

Tetrahedron Lett. 2019, 60, 151226, 10.1016/j.tetlet.2019.151226

Organic synthesis using biocatalysts has been developed over many years and is still a prominent area of research. In this context, various hybrid biocatalysts composed of a synthetic metal complex catalyst and a protein scaffold (i.e. artificial metalloenzymes) have been constructed. One of the most recent research areas in biocatalysts-mediated synthesis is CC bond/cleavage, the most important type of reaction in organic chemistry. Some of the artificial enzymes were applied to in-cell reactions as well as in vitro systems. The effects of the structural fluctuation in biomacromolecules on their functions have also been realized. This review article includes recent research examples of artificial metalloenzymes used to CC bond formation/cleavage. As a perspective, we also focus on how we apply protein dynamics factor for the creation of new generation artificial metalloenzymes.