Construction of artificial metalloenzymes has been a subject of intensive studies due to the utilization of their catalytic ability and functions. There are at least four approaches for the construction of artificial metalloproteins/enzymes; i) site directed mutagenesis, ii) chemical modification of existing proteins/enzymes, iii) introduction of metal cofactors into apo-proteins and iv) use of artificial effector(s) to start the enzymatic reactions. Among them, the latter two approaches are quite novel and Dr. Watanabe has been awarded for his great efforts on these two approaches.
1-1) Preparation of a Pd nano-particle in apo-ferritin: apo-ferritin (apo-Fr) is one of the most popular protein cages as nano reactors to provide unique functions and versatile nano-scale architecture. However, difficulties to utilize the composite materials for chemical reactions have remained yet. Dr. Watanabe prepared a zero-valent palladium cluster by chemical reduction of palladium ions in the apo-Fr cage and examined its catalytic reduction activity. The palladium clusters catalyzes size-selective olefin hydrogenation because substrates must penetrate into the ferritin cavity through the size-restricted channels. Through the Pd⋅apo-Fr study, he has found that there are Pd ion binding sites in the apo-Fr to capture as many as 300 Pd ions. The protein interior surface of apo-Fr contains amino acid residues such as cysteine, histidine, glutamate, aspartate, and methionine. These residues could ligate to metal anions to accommodate them. Thus, the crystal structures of apo-Fr containing various amounts of Pd ions were studied at 1.65Å resolution.

1-2) Pd^II(allyl)⋅apo-ferritin catalyzed Suzuki coupling: Dr. Watanabe prepared various organometallic Pd compounds in the protein cages of apo-Fr and its mutants by their reactions with [Pd^II(allyl)Cl]₂ (allyl =μ³-C3H5). More importantly, he has successfully controlled the coordination structures of these organometallic PdII compounds by replacing histidine residues at the binding sites. A Suzuki coupling reaction of 4-iodoaniline and phenylboronic acid to afford 4-phenylaniline was examined to evaluate the catalytic activities of these composites, since Pd(allyl) complexes are known to catalyze the Suzuki coupling reaction. The turnover number of 3,500 per Pd(allyl)⋅apo-Fr per hour of the coupling reaction was determined by ¹H NMR based on the consumption of 4-iodoaniline and the formation of the product. The size exclusion column chromatography of the reaction solution showed that the spherical 24-mer assembly of the composite is maintained during the reaction.
2) Non-natural substrate oxidation by P450: cytochrome P450s catalyze monooxygenation of organic molecules by consuming expensive NAD(P)H for the reductive activation of molecular oxygen to form the active species, O=Fe(IV) heme^+⋅. Fortunately, there are a couple of P450s which exceptionally utilize H₂O₂ instead of NAD(P)H/O₂ for the monooxygenation such as P450SPα and P450BSβ. Thus, these H₂O₂ dependent P450 are good candidates for bio-catalysts in organic syntesis. Generally speaking, P450s show high substrate specificity for its natural substrates and P450SPα and P450BSβ exclusively oxidize long alkyl chain fatty acids. Thus, first challenge is to provide general methodologies to control its substrate specificity of P450s. So, Dr. Watanabe has developed a series of decoy molecules which allow P450SPα and P450BSβ to oxidize non-natural organic molecules such as styrene, cyclohexane, and propane at reasonable reaction rates. The decoy concept is also applicable to P450BM3, which uses NADPH as the reductant and also exclusively oxidizes long alkyl chain fatty acids. In this particular case, P450BM3 is able to oxidize even ethane to ethanol by using decoy molecules. More importantly, Dr. Watanabe succeeded in the crystal structure analysis of P450BM3/decoy complexes. On the basis of these findings, he has designed a decoy molecule which allow P450BM3 to oxidize methane to methanol.