Expansion of Redox Chemistry in Designer Metalloenzymes

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Expansion of Redox Chemistry in Designer Metalloenzymes Published as part of the Accounts of Chemical Research special issue “Artificial Metalloenzymes and Abiological Catalysis of Metalloenzymes”. Yang Yu,†,§ Xiaohong Liu,‡,§ and Jiangyun Wang*,‡ †

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Department of Biochemical Engineering and Institute for Synthetic Biosystem, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, 5 Zhongguancun South Street, Haidian District, Beijing 100081, China ‡ Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China CONSPECTUS: 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. fine-tune the potentials of redox centers in native metalloenzymes are well established, new methods are still needed for the installation of redox centers in artificial enzymes to dramatically expand their functions. To enhance activities of the designer metalloenzymes, optimizing electron transfer

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nzymes are catalysts for life processes. Designing enzymes from scratch or from an existing scaffold protein could generate artificial enzymes with unprecedented activities1−6 and test our understanding of native enzymes.7−9 Many artificial enzymes catalyze redox reactions using redox-active cofactors or amino acid residues (Figure 1), whose redox potentials typically range from −700 mV to +800 mV (versus standard hydrogen electrode (SHE)).10 While strategies to © XXXX American Chemical Society

Received: December 7, 2018

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Figure 1. Redox potential range of various metal cofactors (black) and genetically encoded UAAs (blue). Most native redox cofactors have redox potentials falling into the range between potentials corresponding to water oxidation and proton reduction to H2 (−400 mV to +800 mV). UAAs could go beyond this range, catalyzing novel redox reactions.

Figure 2. Redox-active UAAs for the expansion of protein redox chemistry. pKa and redox potential values were listed when available. Redox potential (E0) values were measured by pulse radiolysis or cyclic voltammetry; oxidative peak potential (Ep) values were measured by cyclic voltammetry or at pH 7, depending on the compound.

The flow of electrons between redox centers can be described using the semiclassical Marcus equation (eq 1):11

between proteins or between proteins and electrodes or nanomaterials is useful.

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ij π yz zz = jjj 2 j ℏ λk T zz B { k

1/2

EXPANDING REDOX CHEMISTRY WITHIN PROTEIN THROUGH UNNATURAL AMINO ACID (UAA) INCORPORATION Within proteins, redox reactions mainly occur in redox active amino acid residues (Tyr, Trp, Met, Cys, etc., Figure 1) and cofactors (metal ions, metal clusters, or cofactors, flavin, NAD(P)H, etc., Figure 1).10

kET

É ÄÅ ÅÅ (ΔG° + λ)2 ÑÑÑ ÑÑ Å |HAB| expÅÅÅ− Ñ ÅÅÇ 4λkBT ÑÑÑÖ 2

(1)

In this equation, the Gibbs free energy change, ΔG°, is related to the redox potential difference between the redox centers, and the magnitude of the HAB term is dependent on the distance between the two redox centers and their B

DOI: 10.1021/acs.accounts.8b00627 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Accounts of Chemical Research orientation, as well as the medium between them; λ is the reorganization energy for the reaction. Gray and others demonstrated that the Marcus equation can be used to describe the electron transfer process in proteins.12 Redox centers in proteins are often constrained by the protein matrix or redox cofactors leading to a much smaller λ (1 eV). As a result, the key factors for efficient electron transfer in proteins are the redox potential difference, distance, and orientations of the electron donor and acceptor. Many efforts have been dedicated to study how the redox potential of cofactors, especially metal cofactors, can be tuned in native proteins through metal substitution,13 cofactor modification,14 ligand replacement,15 and secondary coordination sphere redesign.16 In contrast, there are only a handful of redox-active amino acids, for which post-translational modifications17 and hydrogen bonding interactions18 have been employed to alter their redox potentials. Such post-translational modifications and strong hydrogen bonds are difficult to design into artificial enzymes. As a result, the redox potentials of amino acid residues cannot be easily altered. Genetic code expansion enables site-specific incorporation of UAAs bearing novel functional groups and chemical properties. Bio-orthogonal reactions, spectroscopic studies, and enzyme design using this technology have been extensively reviewed.19−22 It can also expand the amino acids’ redox chemistry. Tyr residues serve as major electron/proton relays for long-range (>35 Å) proton-coupled electron transfer (PCET) in ribonucleotide reductase. Stubbe, Nocera, and co-workers used Tyr analogues to investigate the remarkable process.23 The redox potentials of 3,4-dihydroxyphenylalanine (DOPA, 1, Figure 2) and 3-aminotyrosine (NH2Y, 2) are lower than that of Tyr, making them an ideal radical trap to locate the residues involved in PCET. In contrast, 3nitrotyrosine (NO2Y, 3) has a higher redox potential than tyrosine, and fluorotyrosines (FnYs, 4−8) have lower pKa values than tyrosine. Replacing the native Tyr residues with these UAAs facilitates the elucidation of the mechanism of the proton-coupled electron transfer (PCET).24 Tyr is also found in many enzyme active sites serving as a redox-active residue. A prominent example is the conserved Tyr residue in the oxygen reduction site of heme copper oxidases (HCOs), which donates an electron and a proton to the O2 substrate during the concerted four-electron/fourproton reduction.25 This unique tyrosine residue forms a crosslink with a nearby histidine residue. Study of a model complex showed that the cross-link lowers the pKa of the phenol group from 10.0 to 8.6.26 The result, along with other studies in model complexes and native enzymes, suggests that the crosslink has a similar function in the native enzyme to facilitate proton transfer.27 Such a post-translational modification (PTM) cannot be easily installed on a protein. Genetic codon expansion shows great potential in studying PTMs, because the technique yields protein with 100% incorporation efficiency at a given site. To directly probe the role of the cross-link in a protein environment, we introduced an UAA, imiTyr (9) into sperm whale myoglobin (Mb), mimicking the His−Tyr cross-link in the native enzyme.8 Mb is chosen as the scaffold protein for the artificial enzyme because it is a simple protein with high expression yield and tolerance to mutations, and it is a natural oxygen binder with high affinity for oxygen. The oxygen reduction activity of the modified protein increased 3-fold to 2.2 min−1 in air-saturated buffer, pH 7.0,

and the mutant was selective for the four-electron/four-proton reduction of O2, producing only 6% reactive oxygen species in the form of H2O2, and the total turnover number of the mutant surpassed 1000. The results confirmed the role of the cross-link in oxidases and points out the possibility to alter the activity of the oxidase by tuning the properties of the phenol group by using UAAs, which we will revisit in this Account. The Tyr−Cys cross-link is another important Tyr posttranslational modification and appears in many metalloenzymes, including galactose oxidase, cysteine dioxygenase, and cytochrome c nitrite reductase.28 Studies of the model compound suggest that such cross-links facilitate the PCET process by lowering the dissociation enthalpy of the phenol O−H bond.28 Using similar design strategy, the posttranslation modification can be mimicked by an UAA, methylthiotyrosine (MtTyr, 12). We incorporated the Tyr− Cys mimicking UAA, into myoglobin through genetic codon expansion. While nitrite reductase catalyzes the six-electron reduction of nitrite to ammonium (eq 2), the mutant enzyme harboring MtTyr in its active site reduces hydroxylamine, a subreaction of nitrite reductase (eq 3), at a rate of 800 min−1, which is 3-fold higher than the rate of the corresponding enzyme harboring a Tyr in its active site.29 These studies suggest that Tyr and its post-translational modifications are critical for enzymatic activity of various redox enzymes. NO2− + 6e− + 8H+ → NH4 + + 2H 2O

(2)

NH 2OH + 2e− + 2H+ → NH4 + + H 2O

(3)

To further explore the role of redox chemistry in the artificial oxidase, we introduced several Tyr analogues into the Mbbased oxidase (3,5-difluorotyrosine, 5; 2,3,5-trifluorotyrosine, 8; 3-chlorotyrosine, 10; 3-methoxytyrosine, 11). These UAAs have oxidative peak potentials spanning from 779 to 847 mV (peak potential at pH 7, Ep for Tyr is 941 mV) and pKa values for the phenol group spanning 6.4 to 9.6 (pKa for Tyr is 10.0).30,31 The reduction potentials of fluorotyrosines were later determined by Tommos and co-workers in a protein scaffold, using protein film square-wave voltammetry.32 Replacing Tyr with its structural analogues causes minimal perturbation to the oxidase, while their oxygen reduction activity is increased from 6.5 mM·min−1 to 15 mM·min−1, correlating to the active site Tyr’s redox potential and pKa. These results suggest that UAA residues with expanded redox properties can be used to modulate the artificial enzyme’s activity in a predictable way.

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EXPANDING REDOX CHEMISTRY IN PROTEIN THROUGH COFACTOR REPLACEMENT Non-native redox cofactors have been incorporated into proteins via rational design and directed evolution. First, non-native redox cofactors with very similar structure to that of the native cofactor can be incorporated into proteins by simply replacing the native cofactor. Hartwig and co-workers introduced metalloporphyrins harboring noble metals, such as iridium, into myoglobin and P450 for efficient carbene transfer reactions.33,34 Such methods require the purification of proteins, extraction of the native cofactor, and reinsertion of the non-native cofactor. Second, mutations can be introduced into proteins such that their binding affinity for the native cofactor is diminished and that for the non-native cofactor enhanced, creating an orthogonal mutant enzyme/non-native cofactor pair. Through this strategy, a mutant enzyme C

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Figure 3. Representative redox cofactors, including heme b, Fe4S4 cluster, copper ion, flavin mononucleotide, and NAD+. UAAs work as high affinity ligands to anchor metal ions in a site selective fashion.

harboring the non-native cofactor can be self-assembled inside the living cell, without the need for a tedious protein purification, refolding, and cofactor insertion process. Brustad and co-workers developed an orthogonal enzyme/unnatural heme pair, where a non-native cofactor, iron deuteroporphyrin IX (Fe-DPIX) (Figure 3) can be selectively incorporated into P450BM3 inside bacterial cells.35 They applied the same facile strategy to incorporate Ir(Me)-deuteroporphyrin IX (Figure 3) into P450 for olefin cyclopropanation.36 To incorporate more complex cofactors, Ward and coworkers developed a supramolecular anchoring approach (Figure 4).6 Taking advantage of the interaction between biotin and (strept)avidin, the strongest noncovalent interaction in nature, they achieved the self-assembly of streptavidin and biotin−transition metal catalyst conjugates in living cells to facilitate unprecedented catalytic reactions, such as olefin metathesis, inside living cells.6 The streptavidin residues that are near the metal binding site strongly influence the efficiency and selectivity of the catalyst, providing a facile approach for improving these artificial metalloenzymes through directed evolution. Despite the successful design of various artificial metalloenzymes harboring a single redox center, the design of artificial enzymes containing two or more redox centers to mimic native bimetallic enzymes remains extremely challenging. Lu and co-workers introduced a 3-His motif into the distal pocket of Mb. The motif can bind the Cu ion, mimicking the CuB site in heme-copper oxidases.38 Recently, the strategy has been expanded from anchoring simple metal ions to a metal cluster. Lu and co-workers redesigned the proximal pocket of cytochrome c peroxidase using Rosetta to accommodate an

Figure 4. Supramolecular anchoring strategy. Reproduced with permission from ref 37. Copyright 2011 American Chemical Society.

Fe4S4 cluster. This Fe4S4 cluster is found in close proximity to the siroheme of native sulfite reductase enzyme and is important for optimal enzymatic activity. After several rounds of rational optimization, the designer metalloenzyme had a kcat of 21.8 min−1, reaching 18% activity of the native sulfite reductase.9 This work established a dependable method for installing complex and functional metal clusters into the scaffold protein, which opens the pathway for rational design and directed evolution. D

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Accounts of Chemical Research Table 1. Distance-Dependent Electron Transfer Rate of UAA-Containing GFP Mutantsa mutant

τ1b [ns] without CuII

τ2b [ns] with CuII

149pyTyr GFP 151pyTyr GFP 182pyTyr GFP 149FNO2Phe GFP 151FNO2Phe GFP 95FNO2Phe GFP 17FNO2Phe GFP

3.4 3.4 3.5

0.7 1.5 2.7

τETc [ps]

distance [Å]

± ± ± ±

8.9 11.4 13.1 8.8 10.3 11.7 12.9

11 73 183 253

0.55 3.5 11 13

kET [s−1] 1.13 0.37 0.08 (9.09 (1.37 (5.46 (3.95

× 109d × 109d × 109d ± 0.45) ± 0.07) ± 0.33) ± 0.20)

× × × ×

1010 1010 109 109

a Reproduced with permission from refs 42 and 44. Copyright 2012 and 2015 American Chemical Society. bFluorescence lifetime of pyTyrincorporated GFP. Photoinduced electron transfer happens between the chromophore and the copper ion. cFluorescence lifetime of FNO2Pheincorporated GFP. Photoinduced electron transfer happens between the chromophore and the UAA. dkET values were calculated from the fluorescence lifetime: kET = τ2−1 (with CuII) − τ2−1 (without CuII).

mV, and there is no redox-active amino acids in the region corresponding to Fe−S clusters or NADPH. We have filled the gap by using a pair of genetically coded UAAs, 3-nitrophenylalanine (NO2Phe, 16) and 4-fluoro-3-nitrophenylalanine (FNO2Phe, 17), with peak potentials of −310 and −470 mV, respectively.44 Data fitting shows the GFP149FNO2Phe mutant having the highest ET rate of (9.09 ± 0.45) × 1010 s−1. This is faster than the ET rate between P700* and A0 in photosystem I.44 To harvest light energy and convert it to chemical energy, we need to create a long-lived excited state and charge separated state, and a redox center with low reduction potential. To achieve these goals, we introduced benzophenone-alanine (Ph2COAla, 18) into the Tyr66 position of superfolder yellow fluorescent protein (sfYFP) and named the resulting protein as photosensitizer protein (PSP). Due to benzophenone’s near 100% quantum efficiency in intersystem crossing from singlet excited state to triplet excited state, the lifetime of the excited state of PSP increases 105 fold versus sfYFP. Importantly, the benzophenone anion radical has a low redox potential (less than −1.14 V), greatly exceeding the lower limit for natural redox centers. Covalently attaching a Niterpyridine complex to PSP enables photocatalyzed CO2 reduction to CO with 2.6% quantum yield, and a TON of 120.45 Direct modification of fluorescent protein’s chromophore using redox and photochemically active UAAs alters their redox and photochemical properties, making the protein an artificial photosynthetic apparatus.

Efficient catalysis requires the design of high-affinity binding sites for redox cofactors, but the task is often challenging. Genetic code expansion enables the integration of multidentate, high affinity ligands for transition metal ion binding. This offers a new approach for redox enzyme design. Roelfes and co-workers used bipyridylalanine (BpyAla, 13) as copper ligand. A single BpyAla is sufficient to facilitate copper ion binding in lactoccocal multidrug resistance regulator (LmrR). The designer enzyme catalyzes Friedel−Crafts alkylation reaction and the hydration of α,β-unsaturated 2-acylpyridine with high enantioselectivity (up to 83% ee and 57% ee, respectively).39,40

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COMBINING UAA AND COFACTOR INCORPORATION: ARTIFICIAL PHOTOSYNTHESIS IN A DESIGNER METALLOENZYME Photosynthesis converts light energy into chemical energy and is a promising solution for sustainable development. Designing artificial photosynthetic apparatus requires recapitulation of nature’s most delicate redox machinery, in which cofactors with optimized redox potentials are aligned. With properly aligned redox centers, electron transfer between P700* and A0 occurs within 10−30 ps: this is the fastest electron transfer process in biological systems.41 To recapitulate the natural system, we engineered green fluorescent protein (GFP) and flavin-containing proteins for enhanced photochemical activity. We introduced two metalchelating amino acids, (S)-2-amino-3-[4-hydroxy-3-(1H-pyrazol-1-yl)phenyl]propanoic acid (pyTyr, 14) and 2-amino-3(8-hydroxyquinolin-5-yl)propanoic acid (HqAla, 15), into proteins.42,43 The pyTyr and HqAla have binding affinity of 3 nM and 0.1 fM for Cu(II), respectively. Such high metal binding affinity makes it possible to anchor metal ions in a specific position in the protein scaffold. UAAs were introduced into different positions in GFP with increasing Cu ion-tofluorophore distance. The GFP mutants showed fluorescence quenching upon Cu(II) binding, and Cu(I) is generated upon photoirradiation, suggesting photoinduced electron transfer in GFP mutants. The electron transfer (ET) rates were calculated based on fluorescence lifetime measurements; they are on the order of nanoseconds and are dependent on the chromophore−metal distance (Table 1). Besides the distances between redox centers, redox potential is another factor affecting the efficiency in photoinduced electron transfer processes. The redox potential of the copper ion in pyTyr-GFP was 168 mV, higher than that of the natural electron acceptor, NADP+.42 As shown in Figure 1, the potentials of generally used UAAs lie between 400 and 800

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ENHANCING ELECTRON TRANSFER USING PROTEIN SELF-ASSEMBLY STRATEGY Besides electron transfer between redox centers in the same protein, electron transfer between proteins is also common in nature. Compared with intraprotein electron transfer processes, interprotein electron transfer involves the formation of an electron transfer complex before the electron transfer step.46 As a result, protein dynamics, such as the formation of the encounter complex or conformational changes upon complex formation, may contribute to the observed electron transfer rate constant. It is often helpful to optimize the steps before the actual electron transfer event for more efficient catalysis. Kd

k3

A ox + Bred HoI A ox /Bred F A red /Box k4

(4)

Directly engineering protein−protein interactions to increase affinity between proteins is an effective strategy for enhancing the ET rate. Myoglobin (Mb) and cytochrome b5 (cyt b5) have weak electrostatic interaction (Ka = 10−3 M−1).47 E

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Accounts of Chemical Research Hoffman and co-workers investigated the interaction between Mb and cyt b5 using Brownian dynamics computation and transient spectroscopy and found the interaction to be weak and dynamic. Three charge-flipping surface mutations on Mb significantly strengthen the electrostatic interaction between Mb and cyt b5 (Figure 5A). The observed kET between Mb

values. For example, direct fusion of P450 monooxygenase CYP79A1 with an electron-transfer protein, ferredoxin, allows electron flow from photosystem I to ferredoxin and then to P450 (Figure 5B).51 The fusion protein showed higher efficiency for the biosynthesis of the cyanogenic glucoside dhurrin, leading to 50% increase in intermediate concentrations in plants with the engineered protein compared to the wild type. Cytochrome P450 is a large protein family carrying out physiologically important reactions. However, P450s’ industrial applications are often limited by the absence of an efficient reductase for P450 activation. There are many attempts at pairing P450s with heterologous reductases52 or using H2O2 as an alternative oxidant to waive the requirement for a reductase.53 Finding a matching, efficient reductase is challenging for researchers. Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) is a heterotrimeric protein complex composed of PCNA1, PCNA2, and PCNA3 with a Kd of 270 nM.54 Fusion of CYP119, putidaredoxin (PdX) and putidaredoxin reductase (PdR) individually to the three components of the PCNA complex could increase the local concentration of reductase to boost electron transfer. As a result, Nagamune and co-workers found that the catalytic activity of the system toward lauric acid hydroxylation is increased 3.5-fold versus the enzymatic systems without PCNA mediated self-assembly.55 PCNA is further used as an anchor to the solid support, and the system showed reusability upon enzyme recycling (Figure 5C).56

Figure 5. Strategies for optimizing interprotein electron transfer through protein self-assembly. (A) Enhancing protein−protein interactions through ionic interactions. (B) Construction of fusion proteins via genetic engineering. (C) Enhancing protein/protein interactions using a scaffold protein.

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ENHANCING PROTEIN REDOX CHEMISTRY USING ELECTRODES AND NANOMATERIALS Compared to homogeneous catalysis, enzymes attached to electrodes can achieve a higher electron transfer rate and can avoid the use of expensive organic reductants. Hydrogenase, nitrogenase, and other natural enzymes have been attached to electrodes for hydrogen production and CO2 reduction.57,58 For example, CO2 reduction has been achieved by Minteer and co-workers, using this approach.58 They attached the vanadium−iron nitrogenase to an electrode. Using cobaltocene as mediator, the system could reduce CO2 to methane or produce ethylene or propene through reductive C−C bond formation at a similar scale. The artificial oxidase designed by Lu and us has shown oxygen reductase activity of 50 s−1, which is close to that of the native enzyme. Dey, Lu, and co-workers wired the same enzyme to a heme covalently attached to the self-assembled monolayer on Au electrode. The catalytic rate reaches 5000 s−1, higher than the native enzyme or synthetic small molecule catalysts on the electrode.59 In hybrid biofuel cells, redox enzymes are displayed on the surface of microorganisms so it is not necessary to purify enzymes. However, due to the limited surface display systems available to researchers, only one or two redox enzymes can be displayed at the anode. The enzymes cannot completely oxidize complex substrates, lowering the total efficiency. Alfonta and co-workers developed the notion of the electrosome, which utilizes the native cellulosome scaffold protein scaffoldin to immobilize alcohol dehydrogenase and formaldehyde dehydrogenase in close proximity. The two redoxactive enzymes form a cascade to oxidize ethanol to acetate, thus enhancing the efficiency of the biofuel cell.60 There are many types of nanomaterials with superb photochemical properties. Combining enzymes with nanomaterials could generate hybrid enzymes. Reisner et al. designed a

with Zn-protoporphyrin and cyt b5 with Fe-protophyrin IX increases from 5.5 × 103 s−1 for the native proteins to 1.0 × 106 s−1.48 Decreasing the negative charge of Mb by using a modified porphyrin molecule further increases interaction between Mb and cyt b5 and raises the kET for charge separation and recombination to 2.1 × 109 and 4.3 × 1010 s−1, respectively. The rate approaches the rate in photosynthetic charge separation (3.3 × 1011 s−1), which is the fastest known in biological systems.48 To increase the electron transfer efficiency between electron donor and acceptor, we introduced cyt b5 and NADH-cyt b5 reductase to shuttle electrons from NADH, the electron donor, to the Mb-based oxidase. The electron transfer cascade was further strengthened by three charge-flipping surface mutations in Mb to enhance the interaction between Mb and cyt b5. The pseudo-first-order rate constant for electron transfer between cyt b5 and G65Y-CuBMb(+6), the Mb-based oxidase with surface mutations for better interaction with cyt b5, is 12 s−1, which is approximately 400-fold faster than that between G65Y-CuBMb and cyt b5. The enhanced electron transfer rate resulted in the same fold of increase in oxygen reduction rate to 50 s−1, making the activity of the artificial enzyme reach the level of the native oxidase.49 Assembled enzymes using scaffold proteins could also be used for enhancing enzyme catalytic efficiency, as reviewed elsewhere.50 Although most enzymes subjected to protein selfassembly design are native enzymes, this strategy is also applicable for artificial enzymes for enhancing redox reactions. Increasing the local concentration of a protein can enhance the protein−protein interactions without changing the Kd F

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Accounts of Chemical Research Biographies

light-driven hydrogenase by covalently attaching the [NiFeSe] hydrogenase from Desulfomicrobium baculatum to Ru-photosensitizer-functionalized TiO2 nanoparticles.61 TiO2 mediates electron transfer between the hydrogenase and photosensitizer, increasing the enzymatic activity by more than 6-fold. In another important demonstration, King and co-workers used cadmium sulfide (CdS) nanocrystals as photosensitizer to drive the reduction of nitrogenase. Importantly, this CdS/nitrogenase hybrid can use light energy to drive the nitrogen reduction reaction, without consuming ATP.62 The hybrid system catalyzes ammonia formation with a TON of 1.1 × 104 over 5 h, a rate comparable to that of the native enzyme. H2 evolution as a byproduct in the hybrid system also suggests that the reaction mechanisms are similar between the CdS/ nitrogenase and nitrogenase/Fe protein systems. By combining redox enzymes with electrodes or nanomaterials, researchers can use biomass to produce electricity or use electricity or light to drive redox reactions to produce useful products, such as hydrogen and hydrocarbons.

Yang Yu received his B.Sc. degree from Peking University in 2008 and his Ph.D. degree from University of Illinois at Urbana−Champaign in 2014 under the direction of Professor Yi Lu. He is an associate professor at School of Chemistry and Chemical Engineering, Beijing Institute of Technology, where he is working on metalloprotein design and engineering. Xiaohong Liu learned physical chemistry at Tianjin normal university, China, and graduated with a B.Sc. degree in 1999. In 2005, she finished her Ph.D. study in organic chemistry at Tsinghua University. Between 2005 and 2008, she worked on telomere shortening evaluation of biologically active nucleosides and nucleotides at Teikyo University of Science and Technology, Japan, as a postdoctoral fellow. Now, she is a professor at Institute of Biophysics, CAS, where she is working on metalloprotein design using genetic code expansion. Jiangyun Wang learned physical chemistry at University of Science and Technology of China and graduated with a B.Sc. degree in 1998. In 2003, he finished his Ph.D. study in metalloprotein chemistry under the supervision of Professor Kenneth Suslick in the University of Illinois at Urbana−Champaign. Between 2003 and 2007, he worked in the laboratory of Professor Peter Schultz at the Scripps Research Institute as a postdoctoral fellow. In 2008, he started his independent lab at the Institute of Biophysics, Chinese Academy of Sciences.

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CONCLUSION This Account reviewed strategies to enhance artificial enzymes’ catalytic activity for redox reactions. Many redox-active UAAs can be incorporated into proteins through genetic codon expansion. Their redox potentials cover the whole range of physiologically relevant potentials. These UAAs are useful for fine-tuning the redox potential in a protein. Moreover, UAAs with strong metal chelating abilities can be used for the precise positioning of metal cofactors for catalysis. Manipulating the position and reduction potential of redox centers in proteins is important to increase the activity of an artificial enzyme. Many designer metalloenzymes need studies to reveal the reaction mechanisms and rate-limiting steps to further increase the activities. One possible route is the electron transfer between proteins, as engineering protein− protein interactions and protein self-assembly using molecular scaffolds can enhance interprotein electron transfer and enzyme activity in many cases. Artificial enzyme design is gaining popularity both in academia and in industry; it is becoming crucial to design functional, as well as efficient, catalysts. We have to admit that cofactor-free enzymes are prevalent because of their low production cost and compatibility for large-scale production. Encouraged by the success of organometallic catalysts, we believe if a designer metalloenzyme is good enough, for example, with a TOF of 100 s−1, a TON of 106, and a reasonable cost, it could solve some real-world problems. The strategies described here should be useful for this purpose.

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ACKNOWLEDGMENTS We are grateful for the financial support from the National Key Research and Development Program of China under Awards 2016YFA0501502, 2017YFA0503704, and 2015CB856203, the National Science Foundation of China under awards 21750003, 91527302, U1632133, and 21878020, Sanming Project of Medicine in Shenzhen (No. SZSM201811092), and the Youth Innovation Promotion Association CAS.

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AUTHOR INFORMATION

Corresponding Author

*[email protected] (J.W.). ORCID

Yang Yu: 0000-0002-0784-9715 Jiangyun Wang: 0000-0001-9748-8898 Author Contributions §

Y.Y. and X.L. contributed equally.

Notes

The authors declare no competing financial interest. G

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