Watanabe, Y.

J. Am. Chem. Soc. 2005, 127, 6556-6562, 10.1021/ja045995q

New methods for the synthesis of artificial metalloenzymes are important for the construction of novel biocatalysts and biomaterials. Recently, we reported new methodology for the synthesis of artificial metalloenzymes by reconstituting apo-myoglobin with metal complexes (Ohashi, M. et al., Angew Chem., Int. Ed.2003, 42, 1005−1008). However, it has been difficult to improve their reactivity, since their crystal structures were not available. In this article, we report the crystal structures of MIII(Schiff base)·apo-A71GMbs (M = Cr and Mn). The structures suggest that the position of the metal complex in apo-Mb is regulated by (i) noncovalent interaction between the ligand and surrounding peptides and (ii) the ligation of the metal ion to proximal histidine (His93). In addition, it is proposed that specific interactions of Ile107 with 3- and 3‘-substituent groups on the salen ligand control the location of the Schiff base ligand in the active site. On the basis of these results, we have successfully controlled the enantioselectivity in the sulfoxidation of thioanisole by changing the size of substituents at the 3 and 3‘ positions. This is the first example of an enantioselective enzymatic reaction regulated by the design of metal complex in the protein active site.

Watanabe, Y.

Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 9416-9421, 10.1073/pnas.0510968103

Protein-to-protein electron transfer (ET) is a critical process in biological chemistry for which fundamental understanding is expected to provide a wealth of applications in biotechnology. Investigations of protein–protein ET systems in reductive activation of artificial cofactors introduced into proteins remains particularly challenging because of the complexity of interactions between the cofactor and the system contributing to ET. In this work, we construct an artificial protein–protein ET system, using heme oxygenase (HO), which is known to catalyze the conversion of heme to biliverdin. HO uses electrons provided from NADPH/cytochrome P450 reductase (CPR) through protein–protein complex formation during the enzymatic reaction. We report that a FeIII(Schiff-base), in the place of the active-site heme prosthetic group of HO, can be reduced by NADPH/CPR. The crystal structure of the Fe(10-CH2CH2COOH-Schiff-base)·HO composite indicates the presence of a hydrogen bond between the propionic acid carboxyl group and Arg-177 of HO. Furthermore, the ET rate from NADPH/CPR to the composite is 3.5-fold faster than that of Fe(Schiff-base)·HO, although the redox potential of Fe(10-CH2CH2COOH-Schiff-base)·HO (−79 mV vs. NHE) is lower than that of Fe(Schiff-base)·HO (+15 mV vs. NHE), where NHE is normal hydrogen electrode. This work describes a synthetic metal complex activated by means of a protein–protein ET system, which has not previously been reported. Moreover, the result suggests the importance of the hydrogen bond for the ET reaction of HO. Our Fe(Schiff-base)·HO composite model system may provide insights with regard to design of ET biosystems for sensors, catalysts, and electronics devices.

Watanabe, Y.

Angew. Chem. Int. Ed. 2003, 42, 1005-1008, 10.1002/anie.200390256

Insertion of a symmetric metal complex, [CrIII(5,5′‐tBu‐salophen)]+ (H2salophen=N,N′‐bis(salicylidene)‐1,2‐phenylenediamine), into the active site of apomyoglobin is demonstrated (see picture). The metal ion and the ligand structure are very important factors that influence the binding affinity of the metal complex with the myoglobin (Mb) cavity. Semisynthetic metalloenzymes can catalyze enantioselective sulfoxidation by using the chiral protein cavity.