Kitagawa, S.; Ueno, T.

Chem. - Asian J. 2014, 9, 1373-1378, 10.1002/asia.201301347

Porous protein crystals, which are protein assemblies in the solid state, have been engineered to form catalytic vessels by the incorporation of organometallic complexes. Ruthenium complexes in cross‐linked porous hen egg white lysozyme (HEWL) crystals catalyzed the enantioselective hydrogen‐transfer reduction of acetophenone derivatives. The crystals accelerated the catalytic reaction and gave different enantiomers based on the crystal form (tetragonal or orthorhombic). This method represents a new approach for the construction of bioinorganic catalysts from protein crystals.

Kitagawa, S.; Ueno, T.

Isr. J. Chem. 2015, 55, 40-50, 10.1002/ijch.201400097

Construction of artificial metalloenzymes based on protein assemblies is a promising strategy for the development of new catalysts, because the three‐dimensional nanostructures of proteins with defined individual sizes can be used as molecular platforms that allow the arrangement of catalytic active centers on their surfaces. Protein needles/tubes/fibers are suitable for supporting various functional molecules, including metal complexes, synthetic molecules, metal nanoparticles, and enzymes with high densities and precise locations. Compared with bulk systems, the protein tube‐ and fiber‐based materials have higher activities for catalytic reactions and electron transfer, as well as enhanced functions when used in electronic devices. The natural and synthetic protein tubes and fibers are constructed by self‐assembly of monomer proteins or peptides. For more precise designs of arrangements of metal complexes, we have developed a new conceptual framework, based on the isolation of a robust needle structure from the cell‐puncturing domains of a bacteriophage. The artificial protein needle shows great promise for use in creating efficient catalytic systems by providing the means to arrange the locations of various metal complexes on the protein surface. In this account, we discuss the recent development of protein needle‐based metalloenzymes, and the future developments we are anticipating in this field.

Arold, S.T.; Eppinger, J.; Groll, M.

ACS Catal. 2019, 9, 11371-11380, 10.1021/acscatal.9b02896

Artificial metalloenzymes (ArMs) have high potential in biotechnological applications as they combine the versatility of transition-metal catalysis with the substrate selectivity of enzymes. An ideal host protein should allow high-yield recombinant expression, display thermal and solvent stability to withstand harsh reaction conditions, lack nonspecific metal-binding residues, and contain a suitable cavity to accommodate the artificial metal site. Moreover, to allow its rational functionalization, the host should provide an intrinsic reporter for metal binding and structural changes, which should be readily amendable to high-resolution structural characterization. Herein, we present the design, characterization, and de novo functionalization of a fluorescent ArM scaffold, named mTFP*, that achieves these characteristics. Fluorescence measurements allowed direct assessment of the scaffold’s structural integrity. Protein X-ray structures and transition metal Förster resonance energy transfer (tmFRET) studies validated the engineered metal coordination sites and provided insights into metal binding dynamics at the atomic level. The implemented active metal centers resulted in ArMs with efficient Diels–Alderase and Friedel–Crafts alkylase activities.