This is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License, which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes. pubs.acs.org/acscatalysis Research Article Cofactor Binding Dynamics Influence the Catalytic Activity and Selectivity of an Artificial Metalloenzyme Lara Villarino,* Shreyans Chordia, Lur Alonso-Cotchico, Eswar Reddem, Zhi Zhou, Andy Mark W. H. Thunnissen, Jean-Didier Mareć hal, and Gerard Roelfes* Cite This: ACS Catal. 2020, 10, 11783−11790 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: We present an artificial metalloenzyme based on the transcriptional regulator LmrR that exhibits dynamics involving the positioning of its abiological metal cofactor. The position of the cofactor, in turn, was found to be related to the preferred catalytic reactivity, which is either the enantioselective Friedel−Crafts alkylation of indoles with β-substituted enones or the tandem Friedel−Crafts alkylation/enantioselective protonation of indoles with α-substituted enones. The artificial metalloenzyme could be specialized for one of these catalytic reactions introducing a single mutation in the protein. The relation between cofactor dynamics and activity and selectivity in catalysis has not been described for natural enzymes and, to date, appears to be particular for artificial metalloenzymes. KEYWORDS: artificial metalloenzymes, biocatalysis, structural dynamics, enzyme design, copper ■ INTRODUCTION Enzymes are remarkable catalysts, capable of catalyzing targets for mutagenesis in the natural- or directed evolution of enzymes, including artificial metalloenzymes, to improve the chemical transformations with high rates and selectivities. A activity.9−18 popular approach to achieve enzymatic catalysis of reactions A special subset of structural dynamics involves the that have no equivalent in nature involves the creation of dynamics in binding of a metal cofactor, that is, the artificial metalloenzymes, which are rationally designed hybrids interchange of cofactor position between multiple cofactor of proteins with abiological catalytically active metal binding sites. This is uncommon in natural enzymes as these cofactors.1−7 In this approach, the basal catalytic activity is usually employ highly evolved cofactor binding sites. However, supplied by the metal complex, whereas the second artificial metalloenzymes are more rudimentary and have not coordination sphere interactions provided by the protein evolved for binding abiological cofactors. Hence, it is possible scaffold are envisioned to contribute to rate acceleration and that the cofactor can bind at multiple sites, that all contribute (enantio-)selectivity. Since the protein scaffolds used have not to the overall catalysis results depending on their relative naturally evolved for the reaction of interest, usually the active abundance and their activity in catalysis. For example, in site structure is far from optimal. artificial metalloenzymes created by dative binding of rhodium One key to the ability of enzymes to accelerate reactions is complexes to apo carbonic anhydrase, the rhodium centers their ability to provide structural complementarity of the active were found to bind at multiple sites in the protein in addition site to the activated complex of the catalyzed reaction by taking advantage of structural dynamics.8 The dynamics of the protein involve conformational changes, e.g., domain rear- Received: April 9, 2020 rangement, loop motions, partial folding/unfolding, etc., Revised: September 11, 2020 crucial to reach the optimal structure of the active site. Published: September 18, 2020 Those flexible regions are also frequently involved in the emergence of alternative active site structures and relate to promiscuous catalytic activities. Thus, they are important © 2020 American Chemical Society https://dx.doi.org/10.1021/acscatal.0c01619 11783 ACS Catal. 2020, 10, 11783−11790 ACS Catalysis pubs.acs.org/acscatalysis Research Article Figure 1. (a) Schematic representation of the self-assembly of the artificial metalloenzyme. (b) Crystal structure of the LmrR/Cu(II)-phen artificial metalloenzyme (PDB: 6R1L). Considerable disorder is observed in the binding mode of Cu-phen, as evidenced by its relatively weakly associated electron density and high atomic B-factors. Disordered ligand binding is a general observation in crystal structures of LmrR and may be an inherent property of this protein. Unfortunately, the weak electron density around the copper, and its special position in the crystal on a crystallographic dyad, prohibited an unambiguous identification of its coordination geometry and ligands other than phenantroline. Close up view of the hydrophobic pore of the LmrR. Residues used in the mutagenesis study are highlighted as sticks (color code as indicated). (c) catalyzed FC reaction (d) catalyzed FC/EP reaction. to the primary metal binding site.19−21 Moreover, cofactor Cu(II)-phen, with a dissociation constant (Kd) of 2.6 ± 2 μM. binding dynamics has been shown to be of key importance in The importance of the central tryptophans for binding Cu(II)- the DNA equivalent of artificial metalloenzymes.22 We have phen is illustrated by the fact that in case of the mutant reported in supramolecular DNA-based catalysis that metal LmrR_W96A, the Kd was 1 order of magnitude lower, i.e., 45 complexes such as copper bipyridine bind reversibly at many μM. different locations in the DNA, resulting in many actives sites The binding of Cu(II)-phen to LmrR was confirmed by X- with differently structured microenvironments in which ray crystallography, which showed the phenanthroline ligand of catalysis occurs with different activity and selectivity.23 As a the complex sandwiched between W96 and W96′, with the result, the overall result of catalysis is the weighted average of indole rings somewhat tilted with respect to each other and the the contribution of all these individual sites.24 Cu(II) complex (Figure 1b). The Cu(II) ion is facing the front Here, we report that dynamics in the binding position of an entrance of the pore. Two carboxylate side chains, from D100 abiological metal cofactor in an artificial metalloenzyme leads and D100′ are oriented toward the Cu(II) ion at a distance of to alternative active site structures in the same protein. We also ∼5 Å and may interact with bound ligands, e.g., water, at the show how the activity and enantioselectivity of this artificial remaining coordination sites at the copper, albeit that these metalloenzyme in two related reactions is dependent on the could not be identified with certainty. Protein residues other position of the metal cofactor in the protein scaffold. Finally, than W96 and D100 that surround the copper within a by a single mutation the artificial metalloenzyme can be distance of 8 Å are predominantly hydrophobic, i.e., V15, A92, specialized toward either of these two catalytic reactions. S97, V99, and I103 (and their equivalents from the dimer ■ RESULTS AND DISCUSSION ARM Assembly and Structure. The design of the artificial mate). Catalysis. In this study, we focused on the application of the artificial enzymes in two catalytic reactions. First, the metalloenzyme is based on the transcription factor Lactococcal previously reported enantioselective vinylogous Friedel−Crafts multidrug resistance Regulator (LmrR), which is a homodi- alkylation of indoles with α, β unsaturated 2-acyl-imidazoles meric protein with a size of 13.5 kDa per monomer that (FC reaction), which gives rise to excellent enantioselectivities, contains an unusually large hydrophobic pore at the dimer to up to 92% ee when catalyzed by LmrR/Cu(II)-phen (Figure interface.25 This hydrophobic pore serves as a promiscuous 1c).28 The second reaction is the tandem Friedel−Crafts binding pocket where planar aromatic molecules bind, as alkylation/enantioselective protonation reaction (FC/EP re- shown in X-ray and NMR structures of LmrR with various action, Figure 1d). It also involves the conjugate addition of planar drugs bound.25−27 Two tryptophan residues, one from indoles to enones, but in this case the substrate carries a each subunit, i.e., W96 and W96′, play a key role in binding by methyl group at the α position and no substituent at the β sandwiching the guest molecule via π-stacking interactions. position. Hence, the chirality is introduced not in the Previously, we have shown that this arrangement is attractive conjugate addition step, but in the subsequent protonation for the supramolecular self-assembly of a novel artificial step. This reaction represents an enantioselective protonation metalloenzyme, by combining LmrR with a Cu(II) complex in water, which is a highly challenging reaction.30 These two with a planar aromatic ligand, like 1,10-phenanthroline (phen; reactions are mechanistically similar, but the chiral center is Figure 1a).28,29 LmrR showed a moderately strong affinity for created in different elementary reaction steps.24 11784 https://dx.doi.org/10.1021/acscatal.0c01619 ACS Catal. 2020, 10, 11783−11790 ACS Catalysis pubs.acs.org/acscatalysis Research Article Table 1. Results of the Tandem Friedel-Crafts/Enantioselective Protonation (FC/EP Reaction) of Indoles with 4a entry indole product catalyst (μM) yield of 5 (%) ee (%)b 1 2b 5b Cu(NO3)2 <5 2 2b 5b Cu(II)-phen <5 3 2b 5b LmrR 10 ± 1 61 ± 2 4 2b 5b Cu(NO3)2 + LmrR 86 ± 10 <5 5 2b 5b Cu(II)-phen + LmrR 58 ± 12 −40 ± 2 6 2a 5a Cu(II)-phen + LmrR 87 ± 14 59 ± 3 a Typical reaction conditions: 4 (1 mM), 2a/b (1 mM) in 20 mM MES buffer (pH 5.0), 150 mM NaCl, at 4 °C for 72h; results are the average of at least two independent experiments, both carried out in duplicate. Error margins represent standard deviations. bdetermined by hplc; + and−signs indicate which is the major enantiomer peak based on order of elution, first and second, respectively. Figure 2. (a) Effect of mutations in LmrR on the ee of the catalyzed FC reaction between 1 and 2a and FC/EP reaction between 4 and 2a. Colors represent the difference between the ΔΔG‡ values calculated from the corresponding ee’s as defined in panel b; (b) visualization of the mutated residues, and the effects on enantioselectivity, onto the crystal structure of LmrR/Cu(II)-phen (metal complex omitted for clarity). The effect of the mutation on the ee, compared to the wild-type LmrR, is visualized as a heatmap where the colors represent the difference between the ΔΔG‡ values calculated from the corresponding ee’s (ΔΔΔG‡ = ΔΔG‡mutant − ΔΔG‡wildtype), indicating an increase (blue) or decrease (red) of enantioselectivity of the reaction catalyzed by the mutant compared to the wild type LmrR. The conjugate addition of 5-methoxy-1H-indole (2b) to 2- that the best results were obtained using 2-methyl-1H-indole methyl-1-(thiazol-2-yl)prop-2-en-1-one (4) was used as bench- (2a), with 87% yield and 59% ee (entry 6) of product 5a. mark FC/EP reaction (Figure 1d, Table 1). The artificial Mutagenesis Studies. Mutagenesis of residues at various metalloenzyme was prepared in situ by self-assembly from 9 positions in the hydrophobic pocket in spatial proximity to mol % of [Cu(phen)(NO3)2] with a slight excess (1.3 equiv) W96 was performed to establish where catalysis of both these of LmrR in MES buffer at pH 5.0. Under these conditions, the reactions occur and which residues are important for activity. product was obtained in 58% yield and 40% ee (entry 5). This included positions at the front entrance, i.e., D100, F93, Reducing the amount of protein to 0.67 equiv, that is, using an and A92, residues in the pocket interior, i.e., M8, Q12, and excess of Cu(II)-phen, gave rise to a similar yield and ee V99 and residues V15, E7, which are placed in the back (Table S5, entry 2 and 4). In absence of catalyst or when using entrance of the pocket. Most of these residues were probed by Cu(II)-phen alone, without protein, almost no reaction was converting them to alanine (alanine scanning), except at observed (entry 1 and 2). Interestingly, using LmrR alone, position A92 were alanine was already present. In this case without Cu(II), gave rise to 10% yield of product with a mutation to glutamate, A92E was performed. significant ee of 61%, albeit of the opposite enantiomer All mutants were evaluated in both the FC reaction of 2- compared to the artificial metalloenzyme (entry 3). As similar methyl-indole (2a) with (E)-1-(1-methyl-1H-imidazol-2-yl)- activity in this reaction was reported recently for the regulatory but-2-en-1-one (1) and the tandem FC/EP reaction of 2a with protein QacR.31 Using a combination of Cu(NO3)2 with LmrR 2-methyl-1-(thiazol-2-yl)prop-2-en-1-one (4). gave good activity, but no ee (entry 4), illustrating the In the FC reaction significant effects on catalysis were importance of the phenanthroline ligand in recruiting the observed in case of the front entrance mutants and the mutant Cu(II) center to the protein. Combined these results show M8A, which is located inside the pore (Figure 2 and Table S6). that, even though protein itself has some activity in this These results confirm that the reaction occurs in the pore, at reaction, it is the combination of a Cu(II)-phen complex and the front entrance, close to the tryptophan residues where the LmrR that is responsible for achieving good catalytic activity Cu(II)-phen complex is bound. In most cases, the effect of the and enantioselectivity. Evaluation of the indole scope showed mutation was negative on both activity and enantioselectivity. 11785 https://dx.doi.org/10.1021/acscatal.0c01619 ACS Catal. 2020, 10, 11783−11790 ACS Catalysis pubs.acs.org/acscatalysis Research Article Two mutations gave rise to significantly improved enantiose- Table 2. Results of the Competitive Binding of Hoechst lectivity: the mutation M8A resulted in a strong increase in the 33342 to LmrR in the Friedel-Crafts (FC Reaction) and the ee to 99% (Table S6, entry 11) and A92E gave rise to both Tandem Friedel-Crafts/Enantioselective Protonation (FC/ complete enantioselectivity (>99% ee) and a significantly EP Reaction)a increased product yield (Table S6, entry 6). As reported before, the W96A mutation caused a significant decrease in both yield and ee (Table S6, entry 2) In contrast, almost all of these mutations had only a small effect on the results of the FC/EP reaction (Figure 2, Table S7). The W96A mutation caused a decrease in the yield, but surprisingly the ee remained similar or was even slightly higher (Table S7, entry 3). The only mutations that gave rise to a significant, negative, effect proved to be Q12E (Table S7, entry 10) and A92E (Table S7, entry 7). For the latter a decrease of Hoechst time the ee to 21% was obtained, which was unexpected since this entry substrate 33342 (eq) (h) product yield (%) ee (%) same mutation proved to be the most beneficial for the FC 1 1 0 72 3a 41 ± 12 92 ± 2 reaction (vide supra). Reducing the amount of protein while 2 1 4 72 3a 29 ± 18 57 ± 5 keeping the Cu(II)-phen concentration the same caused a 3b 4 0 16 5a 53 ± 6 56 ± 1 small increase of the ee to 32% (Table S5, entry 6). 4b 4 4 16 5a 38 ± 1 73 ± 1 Effect of Inhibitors. The role of the hydrophobic pocket in 5b 4 0 72 5a 70 ± 16 58 ± 2 catalysis was probed by inhibition studies using Hoechst 6b 4 4 72 5a 60 ± 6 61 ± 1 H33342, which has been shown to bind with nanomolar a Typical reaction conditions: 1/4 (1 mM), 2a (1 mM), [Cu(II)- affinity in the pocket, sandwiched between W96 and W96′, phen] (9 mol %; 90 μM), LmrR (12 mol %; 120 μM), H33342 (0 or analogous to binding of Cu(II)-phen as observed by X-ray 4 equiv with respect to the concentration of Cu(II)-phen) in 20 mM crystallography.25 MOPS buffer (pH 7.0), 150 mM NaCl, at 4 °C; results are the Fluorescence titration experiments show that H33342 can average of at least two independent experiments, both carried out in indeed displace the Cu(II)-phen from the pore of LmrR, albeit duplicate. b20 mM MES buffer (pH 5.0), 150 mM NaCl. that more than 1 equiv is needed (SI, section V). Interestingly, in the presence of both Cu(II)-Phen and substrate 1 or 4, obtained. In contrast, when we used the mutant LmrR_W96A, resulting in the formation of the substrate bound complexes the FC/EP reaction became the dominant activity, with a Cu(II)-phen -1 and Cu(II)-phen-4, more equivalents of the selectivity of 76%; product 5a was obtained with 22% yield and Hoechst dye are required. This shows that both the substrate 1 69% ee. These results show how with one single mutation, and substrate 4 bound Cu(II) complexes do bind in the pocket either one of these catalyzed reactions can be made the and that this binding is stronger than that of Cu(II)-phen dominant activity of the artificial metalloenzyme. alone (SI, section V). These experiments show that H33342 Effect of A92 Mutation. While the effect of the W96A may act as an inhibitor for catalysis, by displacing Cu(II)-phen mutation is clear, that is, it eliminates a crucial part of the or Cu(II)-phen-substrate complexes from their binding site. In Cu(II)-phen binding site, the role of the glutamate residue in view of the fact that both reactions are protein accelerated, this the A92E mutant was less obvious. The binding affinity of the should have an effect on the results of catalysis. Indeed, [Cu(phen)(NO3)2] was determined (SI, Section VIII) and a addition of increasing amounts of H33342 up to 4 equiv with dissociation constant (Kd) of 65 ± 19 nM and 59 ± 16 nM was respect to Cu(II)-phen caused a significant decrease in the found at pH 7 and 5, respectively. This represents a 2 orders of enantioselectivity in the FC reaction, to 57% ee (Table 2, entry magnitude increase in binding affinity compared to WT LmrR 2). In case of the FC/EP reaction, after 16 h a lower yield was (Table S9). Our initial hypothesis was that the carboxylate observed in the presence of 4 eq of H33342 compared to moieties would contribute to binding of the copper complex by without, as expected. However, the ee was found to be higher interaction with the Cu(II) ion. For this reason, the in the presence of H33342 (Table 2, entries 3 and 4). After 72 corresponding glutamine mutant (i.e., A92Q) was prepared, h, the ee of the reaction in the presence of inhibitor decreased since glutamine is sterically similar to glutamate, but is not a somewhat, which could suggest that H33342 causes good ligand for Cu(II). However, the A92Q mutant also racemization, albeit very slowly (Table 2). showed an increased affinity for Cu(II)-phen (Kd 103 ± 41 Competition Experiments. Next, competition experi- nM; Figure S12), albeit that the results in catalysis were similar ments were performed with the substrates 1 and 4, using wild to the wild-type LmrR (Tables S6, entry 7 and S7, entry 8). type LmrR and the mutants LmrR_W96A and LmrR_A92E The increased affinity of the A92E mutant for Cu(II)-phen also (Figures 3 and S10). When 2-methylindole (2a) was combined allowed to determine the apparent catalytic efficiency of this with equimolar amounts of 1 and 4, the corresponding improved mutant for the FC reaction: kcat/KM = 73.3 M−1 products 3a and 5a were obtained in 66% and 17% yield, min−1 (Figure S14). This information could not be obtained respectively, in the reaction catalyzed by LmrR/Cu(II)-phen, for the wild type LmrR-based artificial metalloenzyme since, in which corresponds to a selectivity of 80% for the FC reaction. this case, the binding affinity of the Cu(II)-phen complex to The enantioselectivity of the products was similar to that the wild type protein is moderate. At higher concentrations obtained in the independent experiments. Notably, when we this causes the substrates to start displacing the Cu(II)-phen carried out these competition experiments with the LmrR complex, which leads to a significant decrease of the catalytic variant A92E, the selectivity for the FC reaction increased to activity.28 Unfortunately, due to substrate solubility issues, the 96%; product 3a was obtained in 66% yield and 98% ee, while individual Michaelis Menten parameters kcat and KM could not only trace amounts of nearly racemic product 5a were be determined. 11786 https://dx.doi.org/10.1021/acscatal.0c01619 ACS Catal. 2020, 10, 11783−11790 ACS Catalysis pubs.acs.org/acscatalysis Research Article Figure 3. (a) Competition experiment between FC reaction of 1 with 2a (and FC/EP reaction of 4 with 2a) catalyzed by LmrR/Cu(II)-phen, LmrR_A92E/Cu(II)-phen and LmrR_W96A/Cu(II)-phen. All substrates were present in equimolar amounts (1 mM). (b) Relative product distribution (%) of the competing FC (green) and FC/EP reactions (blue). (c) ee values for products of the FC and FC/EP reaction in the competition experiment catalyzed by LmrR mutants with standard deviations shown. Computation was then used to gain a better understanding rings of residues W96/W96′ and the phenanthroline ligand are about the effect of mutation A92E. For this purpose, the bis not optimal. Hydrogen bonding interactions between the water aqua form of the copper bound phenanthroline cofactor ligands and residues D100/D100′ contribute to maintain the [Cu(phen)(H2O)2]2+ was optimized via quantum calculations positioning of the copper cofactor. and embedded into the WT, A92E and A92Q variants of LmrR Instead, according to the MD simulations, the mutation of via protein−ligand docking (see details in the Supporting the alanine to glutamate in the A92E mutant disrupts the Information, section X). The best scored structures showed in hydrophobic interaction and generates a hydrogen bonding all cases the copper-phenanthroline moiety at the center of the network at the back of the active site, involving mainly residues cavity packed between tryptophans W96/W96′, a result N88 of helix α4 and N14, located at the opposite side of the consistent with the X-ray structure. Those were subsequently cavity in helix α1 (Figures S16 and S17, left). This contributes submitted to 300 ns MD simulation. to a different structural arrangement of the pore, with a parallel The MD simulations for WT LmrR showed that hydro- orientation of the W96/W96′ indoles and an expanded phobic interactions between A92 and V15 contribute to a hydrophobic free volume of the cavity (Figures S18 and somewhat closed arrangement of the active site, in which the S19). The result is a better packing of the Cu(II)-phen indole rings of W96/W96′ are slightly tilted with respect to complex by a dual π-stacking interaction from W96/W96′. each other, in agreement with the X-ray structure (Figure 4, Tentatively, this is related to the increased binding affinity, and left). Hence, the π-stacking interactions between the indole causes the Cu(II)-phen being positioned deeper inside the pore (Figure S19). While these structures are found frequently in the A92E mutant (40% of the MD simulation), they are virtually nonexistent in case of the WT protein (1% of the MD simulation). Similar structures were observed for the A92Q mutant, albeit these were less frequent than for the A92E mutant (Supporting Information). These results suggest that the A92E mutation does not have a direct effect on catalysis, but mainly has a structural effect, which translates into stronger binding of the Cu(II)-phen complex. Figure 4. Comparison between representative structures of the pore ■ DISCUSSION The both reactions investigated here, the Cu(II)-phen/LmrR along 300 ns of MD simulation for WT (left) and A92E (right) catalyzed FC and FC/EP reaction, are mechanistically similar, variants of LmrR with Cu(II)-phen bound. For WT, hydrophobic the only difference being that in the FC reaction the interactions between A92 and V15 promote closing of the active site, stereocenter is introduced in the conjugate addition step, resulting in a not optimal π-stacking between both W96/W96′ and whereas in the FC/EP reaction the stereocenter is created in the phenanthroline ligand of the Cu(II)-phen cofactor. In contrast, the A92E mutant enables polar interactions with N14, which the subsequent protonation of the enolate intermediate. Yet contribute to the opening of the active site and a parallel orientation some marked differences were observed in how these reactions of the W96/W96′ residues and, thus, cause a better binding of the respond to mutations, addition of inhibitors and in phenanthroline ligand via π-stacking. competition experiments. 11787 https://dx.doi.org/10.1021/acscatal.0c01619 ACS Catal. 2020, 10, 11783−11790 ACS Catalysis pubs.acs.org/acscatalysis Research Article Figure 5. Schematic explanation of the observed metal cofactor binding dynamics of LmrR-based artificial metalloenzymes and the relation to catalysis. Most of the Cu(II)-phen complex will bind in the cavity between the two Trp residues W96/W96′ (primary active site), where it can bind and activate the substrate to undergo conjugate addition by an indole nucleophile, resulting in formation of the product (top pathway, “major species”). This pathway gives rise to the highest ee’s in the FC reaction. However, a small but non-neglible fraction of the Cu(II)-phen can bind at other positions in LmrR (secondary active sites) where it can also activate a substrate for reaction with the indole (lower pathway, “minor” species). This is the pathway that gives the highest ee in the FC/EP reaction. Note that the position of the metal cofactor in the protein in the lower pathway is arbitrary. At present, this secondary site/sites have not been identified. The combined data suggest that the FC and FC/EP reaction favored, such as in the A92E mutant, the enantioselectivity both primarily occur in the hydrophobic pocket of LmrR, with strongly decreases. the Cu(II)-phen catalytic complex bound between the two We propose that this apparent dichotomy can be resolved by central tryptophans W96 and W96′ as suggested by the considering the dynamics of binding of the Cu-phen cofactor, combined spectroscopy and X-ray crystallography data. This is which is a result of the moderate binding affinity of Cu(II)- supported by the fact that removing the tryptophans by phen for wild type LmrR. This means that under the mutation to alanine, or displacing the Cu(II)-phen from the conditions of catalysis, most of the Cu(II)-phen is bound pocket by addition of Hoechst H33342 as inhibitor results in a between the tryptophans, which is the primary active site. decrease of the yield of product in both reactions. Moreover, However, a non-neglible fraction of the Cu(II) complex does the mutagenesis study clearly shows that the ee’s of the FC not bind there and, most likely, can interact at another position reaction are most affected by mutations in the center of the of the protein. At present, the location of this secondary active pore at the front entrance, near W96/W96′, where the Cu(II) site is unknown. It also cannot be ruled out that there is more than one secondary site. These secondary active sites could be site is located in the X-ray structure. Notable in this regard is at a different position in or close to the hydrophobic pore but the A92E mutation, which significantly increases the binding could also be on the surface of the protein (Figure 5). The role affinity of Cu(II)-Phen: this gives rise to the highest activity of the protein is clear as the FC/EP reaction does not occur, or and selectivity in the FC reaction. In the case of the FC/EP only very slowly, in the absence of protein combined with the reaction, the ee’s do not show strong dependence on most of enantioselectivity of product 5. This means that the ARM is the mutations in this location, but it should be noted that the not one well-defined species, but a mixture of copper stereocenter is created in a different elementary step and complexes residing in a different protein microenvironment hence, may not be influenced by mutations in the same way. and the outcome of the catalysis is the weighted average of the However, some of the observations made for the FC/EP contribution of these different active sites. This is highly reaction appear to conflict with the active site being located at reminiscent of what was observed before in our work on the front entrance of the pore and comprised of the Cu(II)- salmon testes DNA-based catalysis.23 phen bound between the W96/W96’. For example, when this In both the FC and FC/EP reaction, the primary active site active site cannot form because Cu(II)-phen binding between dominates the outcome of catalysis because most of the the two tryptophans is blocked, by addition of the inhibitor Cu(II)-phen and the intermediates [Cu(II)-phen-1] and Hoechst H33342, or by removal of W96/W96’ by muta- [Cu(II)-phen-2] are bound here and it significantly accelerates genesis, the enantioselectivity of the reaction actually increases. the reaction. This is also the reason that, when using an excess Conversely, in cases where the binding of Cu(II)-phen is of Cu(II)-phen compared to protein, the results of catalysis do 11788 https://dx.doi.org/10.1021/acscatal.0c01619 ACS Catal. 2020, 10, 11783−11790 ACS Catalysis ■ pubs.acs.org/acscatalysis Research Article not decrease. In case of the FC reaction this also gives the AUTHOR INFORMATION highest enantioselectivity. But in case of the FC/EP reaction, it Corresponding Authors appears that the primary site is not the most enantioselective, but the secondary site(s). Indeed by blocking or eliminating Lara Villarino − Stratingh Institute for Chemistry, University of the primary active site by removal of W96, the relative Groningen, 9747, AG, Groningen, The Netherlands; contribution of secondary site(s) increases, resulting in a orcid.org/0000-0003-4728-2001; Email: lara.villarino@ higher ee. Conversely, by favoring the primary active site usc.es through the A92E mutation, which strongly increases the Gerard Roelfes − Stratingh Institute for Chemistry, University of affinity of Cu(II)-phen for binding between W96/W96′, Groningen, 9747, AG, Groningen, The Netherlands; basically there is/are no more secondary site(s), resulting in orcid.org/0000-0002-0364-9564; Email: j.g.roelfes@rug.nl a decreased enantioselectivity in the FC/EP reaction but an Authors increase in the FC reaction. By using an excess of Cu(II)-phen compared to LmrR_A92E mutant, some of the secondary sites Shreyans Chordia − Stratingh Institute for Chemistry, get occupied again, which is reflected in an small increase of ee University of Groningen, 9747, AG, Groningen, The in the case of the FC/EP reaction. Netherlands The competition experiments are in agreement with this Lur Alonso-Cotchico − Stratingh Institute for Chemistry, hypothesis. Using LmrR, both the FC and FC/EP reaction are University of Groningen, 9747, AG, Groningen, The possible, but the former is preferred in the primary active site. Netherlands; orcid.org/0000-0002-0172-6394 Making a single mutation, A92E, results in further favoring of Eswar Reddem − Stratingh Institute for Chemistry, University of reaction via the primary active site (top pathway) and the Groningen, 9747, AG, Groningen, The Netherlands secondary site(s) (lower pathway) is effectively shut down Zhi Zhou − Stratingh Institute for Chemistry, University of (Figure 5). This is reflected in the results: the A92E mutant Groningen, 9747, AG, Groningen, The Netherlands; strongly prefers the FC reaction over the FC/EP reaction. orcid.org/0000-0001-7926-118X Conversely, by removing the central tryptophans via the W96A Andy Mark W. H. Thunnissen − Groningen Biomolecular mutation only the lower pathway can be followed, since it Sciences and Biotechnology Institute, University of Groningen, eliminates the preferred primary binding site for the Cu(II)- 9747, AG, Groningen, The Netherlands Jean-Didier Maréchal − Departament de Quimica, ́ Universitat phen complex. The FC reaction is still possible, albeit with lower activity and selectivity. In this case, the FC/EP reaction Autònoma de Barcelona, 08193 Barcelona, Spain; is now the favored reaction, resulting in an increased yield and orcid.org/0000-0002-8344-9043 enantioselectivity of the FC/EP product. Complete contact information is available at: ■ CONCLUSION In conclusion, the LmrR/Cu(II)-phen artificial metalloenzyme https://pubs.acs.org/10.1021/acscatal.0c01619 Notes shows dynamic behavior in the positioning of its abiological The authors declare no competing financial interest. metal cofactor, which, in turn, is related to the activity and selectivity in catalysis. In the WT LmrR/Cu(II)-phen, the FC reaction, which occurs in the primary active site in the ■ ACKNOWLEDGMENTS We acknowledge the European Synchrotron Radiation Facility hydrophopbic pore of the protein, is the preferred activity. for provision of synchrotron radiation facilities. The authors However, it exhibits lower, but significant, levels of activity for thank R. Leveson-Gower for assistance in catalysis experi- another reaction, the FC/EP reaction, which is mechanistically ments. This project was supported by the European Research similar but involves an enantioselective protonation step. Council (ERC starting Grant No. 280010), The Netherlands However, the FC/EP reaction achieves the highest enantiose- Organisation for Scientific Research (NWO; Vici Grant lectivity when it occurs in a secondary active site. By only one 724.013.003), a postdoctoral grant from Xunta de Galicia mutation, A92E, this artificial metalloenzyme became almost (Plan I2C, to L.V.), and a grant from the Spanish MINECO fully selective for the FC reaction, while by another mutation, (CTQ2017-87889-P). G.R. acknowledges support from the i.e., W96A, the FC/EP reaction became the dominant activity. Ministry of Education Culture and Science (Gravitation The switching of preferred activity and selectivity by dynamic Programme No. 024.001.035). ■ interconversion of the position of a metal cofactor has not been described for natural enzymes and, to date, appears to be particular for artificial metalloenzymes. 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