Lu, Y.; Wang, J.

J. Am. Chem. Soc. 2015, 137, 11570-11573, 10.1021/jacs.5b07119

Terminal oxidases catalyze four-electron reduction of oxygen to water, and the energy harvested is utilized to drive the synthesis of adenosine triphosphate. While much effort has been made to design a catalyst mimicking the function of terminal oxidases, most biomimetic catalysts have much lower activity than native oxidases. Herein we report a designed oxidase in myoglobin with an O2 reduction rate (52 s–1) comparable to that of a native cytochrome (cyt) cbb3 oxidase (50 s–1) under identical conditions. We achieved this goal by engineering more favorable electrostatic interactions between a functional oxidase model designed in sperm whale myoglobin and its native redox partner, cyt b5, resulting in a 400-fold electron transfer (ET) rate enhancement. Achieving high activity equivalent to that of native enzymes in a designed metalloenzyme offers deeper insight into the roles of tunable processes such as ET in oxidase activity and enzymatic function and may extend into applications such as more efficient oxygen reduction reaction catalysts for biofuel cells.

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.

Lu, Y.; Wang, J.

Chem. Sci. 2015, 6, 3881-3885, 10.1039/C5SC01126D

While nature employs various covalent and non-covalent strategies to modulate tyrosine (Y) redox potential and pKa in order to optimize enzyme activities, such approaches have not been systematically applied for the design of functional metalloproteins. Through the genetic incorporation of 3-methoxytyrosine (OMeY) into myoglobin, we replicated important features of cytochrome c oxidase (CcO) in this small soluble protein, which exhibits selective O2 reduction activity while generating a small amount of reactive oxygen species (ROS). These results demonstrate that the electron donating ability of a tyrosine residue in the active site is important for CcO function. Moreover, we elucidated the structural basis for the genetic incorporation of OMeY into proteins by solving the X-ray structure of OMeY specific aminoacyl-tRNA synthetase complexed with OMeY.