Woon Ju Song

The generation of new enzymatic activities has mainly relied on repurposing the interiors of preexisting protein folds because of the challenge in designing functional, three-dimensional protein structures from first principles. Here we report an artificial metallo-β-lactamase, constructed via the self-assembly of a structurally and functionally unrelated, monomeric redox protein into a tetrameric assembly that possesses catalytic zinc sites in its interfaces. The designed metallo-β-lactamase is

The reactions of manganese(III) porphyrin complexes with terminal oxidants, such as m-chloroperbenzoic acid, iodosylarenes, and H(2)O(2), produced high-valent manganese(V)-oxo porphyrins in the presence of base in organic solvents at room temperature. The manganese(V)-oxo porphyrins have been characterized with various spectroscopic techniques, including UV-vis, EPR, 1H and 19F NMR, resonance Raman, and X-ray absorption spectroscopy. The combined spectroscopic results indicate that the manganese

From the catalytic reactions that sustain the global oxygen, nitrogen, and carbon cycles to the stabilization of DNA processing proteins, transition metal ions and metallocofactors play key roles in biology. Although the exquisite interplay between metal ions and protein scaffolds has been studied extensively, the fact that the biological roles of the metals often stem from their placement in the interfaces between proteins and protein subunits is not always recognized. Interfacial metal ions st

We have shown previously that iodosylbenzene-iron(III) porphyrin intermediates (2) are generated in the reactions of oxoiron(IV) porphyrin pi-cation radicals (1) and iodobenzene (PhI), that 1 and 2 are at equilibrium in the presence of PhI, and that the epoxidation of olefins by 2 affords high yields of epoxide products. In the present work, we report detailed mechanistic studies on the nature of the equilibrium between 1 and 2 in the presence of iodoarenes (ArI), the determination of reactive s

We describe the design and evolution of catalytic hydrolase activity on a supramolecular protein scaffold, Zn₄:^C96RIDC1₄, which was constructed from cytochrome cb₅₆₂ building blocks via a metal-templating strategy. Previously, we reported that Zn₄:^C96RIDC1₄ could be tailored with tripodal (His/His/Glu), unsaturated Zn coordination motifs in its interfaces to generate a variant termed Zn₈:^A104AB3<su

By combining synthetic catalysts and biochemical tools, numerous artificial metalloenzymes have been designed to exhibit high catalytic activity and selectivity in diverse chemical transformations. Out of the nearly infinite number of discovered or characterised proteins, however, only a handful of proteins have been employed as scaffolds for artificial metalloenzymes, implying that specific proteins are preferred owing to their native structural, functional, or biochemical properties. In the pr

Toluene/o-xylene monooxygenase hydroxylase (ToMOH), a diiron-containing enzyme, can activate dioxygen to oxidize aromatic substrates. To elucidate the role of a strictly conserved T201 residue during dioxygen activation of the enzyme, T201S, T201G, T201C, and T201V variants of ToMOH were prepared by site-directed mutagenesis. X-ray crystal structures of all the variants were obtained. Steady-state activity, regiospecificity, and single-turnover yields were also determined for the T201 mutants. D

We report the observation of a novel intermediate in the reaction of a reduced toluene/o-xylene monooxygenase hydroxylase (ToMOH(red)) T201S variant, in the presence of a regulatory protein (ToMOD), with dioxygen. This species is the first oxygenated intermediate with an optical band in any toluene monooxygenase. The UV-vis and Mossbauer spectroscopic properties of the intermediate allow us to assign it as a peroxodiiron(III) species, T201S(peroxo), similar to H(peroxo) in methane monooxygenase.

Efficient and environmentally friendly conversion of light energy for direct utilization in chemical production has been a long-standing goal in enzyme design. Herein, we synthesized artificial photocatalytic enzymes by introducing an Ir photocatalyst and a Ni(bpy) complex to an optimal protein scaffold in close proximity. Consequently, the enzyme generated C-O coupling products with up to 96% yields by harvesting visible light and performing intramolecular electron transfer between the two cata

For numerous enzymes reactive toward small gaseous compounds, growing evidence indicates that these substrates diffuse into active site pockets through defined pathways in the protein matrix. Toluene/o-xylene monooxygenase hydroxylase is a dioxygen-activating enzyme. Structural analysis suggests two possible pathways for dioxygen access through the α-subunit to the diiron center: a channel or a series of hydrophobic cavities. To distinguish which is utilized as the O(2) migration pathway, the di

Site-directed mutagenesis studies of a strictly conserved T201 residue in the active site of toluene/o-xylene monooxygenase hydroxylase (ToMOH) revealed that a single mutation can facilitate kinetic isolation of two distinctive peroxodiiron(III) species, designated T201(peroxo) and ToMOH(peroxo), during dioxygen activation. Previously, we characterized both oxygenated intermediates by UV-vis and Mössbauer spectroscopy, proposed structures from DFT and QM/MM computational studies, and elucidated

The catalytic functions of metalloenzymes are often strongly correlated with metal elements in the active sites. However, dioxygen-activating nonheme quercetin dioxygenases (QueD) are found with various first-row transition-metal ions when metal swapping inactivates their innate catalytic activity. To unveil the molecular basis of this seemingly promiscuous yet metal-specific enzyme, we transformed manganese-dependent QueD into a nickel-dependent enzyme by sequence- and structure-based directed

ConspectusMetalloproteins establish a comprehensive molecular space by combining inorganic cofactors with protein environments. The chemical interplay between a metal element and a protein matrix is remarkable yet elusive, as the chemical properties of metal ions do not directly translate into those of metalloenzymes when placed within a protein matrix. Instead, the biochemical context determines the metal-coordination geometries, reaction kinetics, and thermodynamic parameters, such as redox po

Artificial metalloenzymes have fed our understanding of how inorganic reactivities emerge, evolve, and diversify in protein environments. Herein, we created dinuclear copper oxidases by genetically encoding a metal-ligating unnatural amino acid (bpy-Ala) per protomer in the vicinity of the innate C₂ rotational axis of a homo-oligomeric protein. The inherent protein symmetry allows the precise multiplication and placement of two Cu(bpy) species. Depending on the location of bpy-Ala, th