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.

Wang, J.

ACS Catal. 2018, 8, 1851-1863, 10.1021/acscatal.7b03754

There are 20 proteinogenic amino acids and a limited number of cofactors naturally available to build enzymes. Genetic codon expansion enables us to incorporate more than 200 unnatural amino acids into proteins using cell translation machinery, greatly expanding structures available to protein chemists. Such tools enable scientists to mimic the active site of an enzyme to tune enzymatic activity, anchor cofactors, and immobilize enzymes on electrode surfaces. Non-native cofactors can be incorporated into the protein through covalent or noncovalent interactions, expanding the reaction scope of existing enzymes. The review discusses strategies to incorporate unnatural amino acids and non-native cofactors and their applications in tuning and expanding enzymatic activities of artificial metalloenzymes.

Liu, X.; Wang, J.; Wu, Y.; Zhong, F.

J. Am. Chem. Soc. 2021, 143, 617-622, 10.1021/jacs.0c10882

Devising artificial photoenzymes for abiological bond-forming reactions is of high synthetic value but also a tremendous challenge. Disclosed herein is the first photobiocatalytic cross-coupling of aryl halides enabled by a designer artificial dehalogenase, which features a genetically encoded benzophenone chromophore and site-specifically modified synthetic NiII(bpy) cofactor with tunable proximity to streamline the dual catalysis. Transient absorption studies suggest the likelihood of energy transfer activation in the elementary organometallic event. This design strategy is viable to significantly expand the catalytic repertoire of artificial photoenzymes for useful organic transformations.

Lu, Y.; Wang, J.

J. Am. Chem. Soc. 2015, 137, 4594-4597, 10.1021/ja5109936

While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pKa and reduction potential in O2 reduction have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O2 reduction mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pKa of the phenol ring and increasing reduction potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O2 reduction activity and the fraction of H2O formation with the pKa of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H2O2, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivatives incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymatic activity beyond that of natural Tyr.

Wang, J.

Acc. Chem. Res. 2019, 52, 557-565, 10.1021/acs.accounts.8b00627

Many artificial enzymes that catalyze redox reactions have important energy, environmental, and medical applications. Native metalloenzymes use a set of redox-active amino acids and cofactors as redox centers, with a potential range between −700 and +800 mV versus standard hydrogen electrode (SHE, all reduction potentials are versus SHE). The redox potentials and the orientation of redox centers in native metalloproteins are optimal for their redox chemistry. However, the limited number and potential range of native redox centers challenge the design and optimization of novel redox chemistry in metalloenzymes. Artificial metalloenzymes use non-native redox centers and could go far beyond the natural range of redox potentials for novel redox chemistry. In addition to designing protein monomers, strategies for increasing the electron transfer rate in self-assembled protein complexes and protein–electrode or −nanomaterial interfaces will be discussed. Redox reactions in proteins occur on redox active amino acid residues (Tyr, Trp, Met, Cys, etc.) and cofactors (iron sulfur clusters, flavin, heme, etc.). The redox potential of these redox centers cover a ∼1.5 V range and is optimized for their specific functions. Despite recent progress, tuning the redox potential for amino acid residues or cofactors remains challenging. Many redox-active unnatural amino acids (UAAs) can be incorporated into protein via genetic codon expansion. Their redox potentials extend the range of physiologically relevant potentials. Indeed, installing new redox cofactors with fined-tuned redox potentials is essential for designing novel redox enzymes. By combining UAA and redox cofactor incorporation, we harnessed light energy to reduce CO2 in a fluorescent protein, mimicking photosynthetic apparatus in nature. Manipulating the position and reduction potential of redox centers inside proteins is important for optimizing the electron transfer rate and the activity of artificial enzymes. Learning from the native electron transfer complex, protein–protein interactions can be enhanced by increasing the electrostatic interaction between proteins. An artificial oxidase showed close to native enzyme activity with optimized interaction with electron transfer partner and increased electron transfer efficiency. In addition to the de novo design of protein–protein interaction, protein self-assembly methods using scaffolds, such as proliferating cell nuclear antigen, to efficiently anchor enzymes and their redox partners. The self-assembly process enhances electron transfer efficiency and enzyme activity by bringing redox centers into close proximity of each other. In addition to protein self-assembly, protein–electrode or protein–nanomaterial self-assembly can also promote efficient electron transfer from inorganic materials to enzyme active sites. Such hybrid systems combine the efficiency of enzyme reactions and the robustness of electrodes or nanomaterials, often with advantageous catalytic activities. By combining these strategies, we can not only mimic some of nature’s most fascinating reactions, such as photosynthesis and aerobic respiration, but also transcend nature toward environmental, energy, and health applications.

Liu, X.; Tian, C.; Wang, J.

ACS Catal. 2021, 11, 5628-5635, 10.1021/acscatal.1c00287

Photosystem I (PSI) is a very large membrane protein complex (∼1000 kDa) harboring P700*, the strongest reductant known in biological systems, which is responsible for driving NAD(P)+ and ultimately for CO2 reduction. Although PSI is one of the most important components in the photosynthesis machinery, it has remained difficult to enhance PSI functions through genetic engineering due to its enormous complexity. Inspired by PSI’s ability to undergo multiple-step photo-induced electron hopping from P700* to iron–sulfur [Fe4S4] clusters, we designed a 33 kDa miniature photocatalytic CO2-reducing enzyme (mPCE) harboring a chromophore (BpC) and two [Fe4S4] clusters (FeA/FeB). Through reduction potential fine-tuning, we optimized the multiple-step electron hopping from BpC to FeA/FeB, culminating in a CO2/HCOOH conversion quantum efficiency of 1.43%. As mPCE can be overexpressed with a high yield in Escherichia coli cells without requiring synthetic cofactors, further development along this route may result in rapid photo-enzyme quantum yield improvement and functional expansion through an efficient directed evolution process.

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.

Lu, Y.; Wang, J.

Angew. Chem. Int. Ed. 2012, 51, 4312-4316, 10.1002/anie.201108756

Top model: Heme–copper oxidase (HCO) contains a histidine–tyrosine cross‐link in its heme a3/CuB oxygen reduction center. A functional model of HCO was obtained through the genetic incorporation of the unnatural amino acid imiTyr, which mimics the Tyr–His cross‐link, and of the CuB site into myoglobin (see picture). Like HCO, this small soluble protein exhibits selective O2‐reduction activity while generating little reactive oxygen species.