Hemoproteins Reconstituted with Artificial Metal Complexes as

2 days ago - Biography. Koji Oohora received his Ph.D. degree from Osaka University in 2011. He has been an Assistant Professor at Osaka University ...
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Hemoproteins Reconstituted with Artificial Metal Complexes as Biohybrid Catalysts Published as part of the Accounts of Chemical Research special issue “Artificial Metalloenzymes and Abiological Catalysis of Metalloenzymes”. Koji Oohora, Akira Onoda, and Takashi Hayashi*

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Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan CONSPECTUS: In nature, heme cofactor-containing proteins participate not only in electron transfer and O2 storage and transport but also in biosynthesis and degradation. The simplest and representative cofactor, heme b, is bound within the heme pocket via noncovalent interaction in many hemoproteins, suggesting that the cofactor is removable from the protein, leaving a unique cavity. Since the cavity functions as a coordination sphere for heme, it is of particular interest to investigate replacement of native heme with an artificial metal complex, because the substituted metal complex will be stabilized in the heme pocket while providing alternative chemical properties. Thus, cofactor substitution has great potential for engineering of hemoproteins with alternative functions. For these studies, myoglobin has been a focus of our investigations, because it is a well-known oxygen storage hemoprotein. However, the heme pocket of myoglobin has been only arranged for stabilizing the heme-bound dioxygen, so the structure is not suitable for activation of small molecules such as H2O2 and O2 as well as for binding an external substrate. Thus, the conversion of myoglobin to an enzyme-like biocatalyst has presented significant challenges. The results of our investigations have provided useful information for chemists and biologists. Our own efforts to develop functionalized myoglobin have focused on the incorporation of a chemically modified cofactor into apomyoglobin in order to (1) construct an artificial substrate-binding site near the heme pocket, (2) increase cofactor reactivity, or (3) promote a new reaction that has never before been catalyzed by a native heme enzyme. In pursuing these objectives, we first found that myoglobin reconstituted with heme having a chemically modified heme-propionate side chain at the exit of the heme pocket has peroxidase activity with respect to oxidation of phenol derivatives. Our recent investigations have succeeded in enhancing oxidation and oxygenation activities of myoglobin as well as promoting new reactions by reconstitution of myoglobin with new porphyrinoid metal complexes. Incorporation of suitable metal porphyrinoids into the heme pocket has produced artificial enzymes capable of efficiently generating reactive high valent metal−oxo and metallocarbene intermediates to achieve the catalytic hydroxylation of C(sp3)−H bonds and cyclopropanation of olefin molecules, respectively. In other efforts, we have focused on nitrobindin, an NO-binding hemoprotein, because aponitrobindin includes a β-barrel cavity, which provides a robust structure highly similar to that of the native holoprotein. It was expected that the aponitrobindin would be suitable for development as a protein scaffold for a metal complex. Recently, it was confirmed that several organometallic complexes can bind to this scaffold and function as catalysts promoting hydrogen evolution or C−C bond formation. The hydrophobic β-barrel structure plays a significant role in substrate binding as well as controlling the stereoselectivity of the reactions. Furthermore, these catalytic activities and stereoselectivities are remarkably improved by mutation-dependent modifications of the cavity structure for the artificial cofactor. This Account demonstrates how apoproteins of hemoproteins can provide useful protein scaffolds for metal complexes. Further development of these concepts will provide a useful strategy for generation of robust and useful artificial metalloenzymes.

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INTRODUCTION

major heme cofactor being known as heme b, Fe protoporphyrin IX (1), is noncovalently bound within the protein via coordination, hydrogen bonding, and hydrophobic and π−π interactions. For example, heme b is employed as the heme cofactor in myoglobin, hemoglobin, cytochrome P450s, horseradish peroxidase, and cytochrome b5, among others. Therefore,

Hemoproteins, which use iron porphyrin (heme) as a cofactor, are among the most versatile metalloproteins. The heme cofactor works as a reaction center to provide biochemical functions ranging from O2 or NO storage and transport to catalysis and electron transfer.1 These functions are mainly derived from the unique arrangements between the cofactor and protein matrices formed by the heme pocket. Several different versions of heme cofactors exist in nature, and the simplest © XXXX American Chemical Society

Received: December 31, 2018

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Figure 1. Representative scheme of hemoprotein reconstitution.

2: substitution of the central metal atom, modification of peripheral side chains, and replacement of the porphyrin

these proteins are converted to corresponding apoproteins after removal of heme under acidic conditions as shown in Figure 1.2 Furthermore, addition of native hemin, ferric 1, into the apoprotein results in reconstituted protein, which has physicochemical properties and reactivity essentially identical to those of the original protein.3 These reconstitution processes are well-known, and several research groups have prepared hemoproteins reconstituted with modified heme cofactors. Their aim has been the evaluation of the interactions between heme and the heme pocket, which often works as a second coordination sphere.4 In contrast, our recent work has focused on reconstitution to engineer hemoprotein functions using heme-propionate modified hemes as well as non-native metalloporphyrinoids.4,5 In one such example, we reported that myoglobin reconstituted with iron porphycene significantly enhances O2 affinity with opposing O2/CO discrimination relative to native myoglobin (nMb).6 This investigation of modified heme structure significantly improved our understanding of the structure− function relationship of hemoproteins and provided insights into construction of new biomaterials and devices. Accordingly, we have been engaged in efforts to combine an artificial metal cofactor with an apoprotein after removal of native heme from the hemoprotein because the apoprotein provides a unique vacant heme pocket that can be employed as a useful cavity capable of forming an effective coordination sphere of artificially created metalloenzymes.7,8 In our recent investigations, apomyoglobin, apo-horseradish peroxidase, apo-cytochrome P450cam, apo-cytochrome b562, apo-cytochrome c, and aponitrobindin have been employed as protein scaffolds to generate artificial metalloenzymes capable of extending the scope of reactivity of the native proteins with excellent activity and selectivity. We refer to these artificial metalloenzymes as biohybrid catalysts.

Figure 2. Molecular structure of protoporphyrin IX iron complex (1) (heme b) and three different types of heme modification.

framework with artificial porphyrinoid framework. According to this strategy, we have prepared several artificially created cofactors, which can be grouped into three categories as shown in Figure 3. In category 1, the heme-propionate side chains are modified to obtain a functionalized cofactor. The side chains are generally located near the exit of the heme pocket, thereby allowing them to provide an interface or mediator with an external substrate. Category 2 includes a group of cofactors with an artificial metalloporphyrinoid consisting of a tetrapyrrole macrocycle ligand such as porphycene or corrole. Cofactors that belong to category 3 are completely different from the metalloporphyrinoids. Representative cofactors are organometallic species that have activities toward organic reactions not seen in hemoproteins.

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ENGINEERING OF HEMOPROTEINS BY COFACTOR SUBSTITUTION Apoprotein is typically obtained upon addition of HClaq in a hemoprotein solution. After acid denaturation, 2-butanone is added to extract native heme from the mixture while the apoprotein remains in the water layer. Neutralization and subsequent lyophilization of the aqueous layer provide white powder of the corresponding apoprotein, which is substantially free of heme, as evidenced by a lack of characteristic porphyrin absorption bands in a UV−visible absorption spectrum.3 It is known that several heme b-based hemoproteins can be converted into the corresponding apoproteins. Gentle dropwise addition of a cofactor into a solution of apoprotein yields the corresponding reconstituted hemoprotein. To engineer hemoproteins using reconstitution methods, our group has prepared various artificial cofactors as shown in Figure

HEME-PROPIONATE MODIFIED COFACTORS

First Trial

Our research group began converting myoglobin into an artificial enzyme via insertion of a metal complex into apomyoglobin in the 1990s. Myoglobin is an oxygen storage hemoprotein with a well-known and relatively stable 3D structure. It does not have the same natural peroxidase activity exhibited by the heme-dependent peroxidases. There are two reasons for this functional behavior: (i) while H2O2 binds to heme in myoglobin, it is not properly activated to provide peroxidase activity and (ii) there is no obvious substrate-binding domain in myoglobin. To enable myoglobin to promote H2O2dependent oxidation of phenol derivatives, we first focused on the latter and introduced benzene moieties to the terminus of each heme-propionate side chain to construct a hydrophobic pocket at the entrance of the heme pocket (Figure 4).9 The UV−vis titrimetric measurement of 2-methoxyphenol (guaiaB

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Figure 3. Examples of molecular structures of artificial cofactors. The incorporation of these cofactors into an apoprotein can be confirmed by UV−vis, CD, or mass spectroscopic methods.

azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and catechol, among others. During the same period, several other research groups also demonstrated enzymatic activities of other examples of heme-propionate modified reconstituted myoglobin.10−12 Peroxidation

As mentioned above, modification of the heme-propionate side chain is an effective way to increase the peroxidase activity of myoglobin. However, this enhanced activity is still lower than the peroxidase activity provided by native horseradish peroxidase (nHRP), for example. To improve the peroxidase activity of the previously described reconstituted myoglobin and convert myoglobin to a peroxidase-like biocatalyst, our group modified the heme pocket as well as the cofactor, since myoglobin mutants where His64 in the heme pocket is replaced with Ala or Asp are known to have increased peroxidase activity.13 A Michaelis−Menten kinetics analysis demonstrated that the Km value for the H64A and H64D mutants with heme 2, rMbH64A(2) and rMbH64D(2), clearly decreases, and the kcat/Km value increases for H2O2-depedent guaiacol oxidation. Furthermore, optimization of the cofactor structure finally provided remarkably enhanced peroxidase activity for rMbH64D(3) where heme 3 was inserted into the H64D mutant.14 In fact, the Michaelis−Menten kinetics analysis indicates that the activity of rMbH64D(3) is near the level of the peroxidase activity of nHRP (Table 1).15 These results indicate that although the natural

Figure 4. Plausible structure of rMb(2) with a hydrophobic domain at the entrance of the heme pocket.

col) in a solution of myoglobin reconstituted with heme 2, rMb(2), provided significant spectral changes of the Soret band with a Kd value of 0.083 mM at 22 °C, whereas no spectral changes were observed for nMb treated in a similar manner. This finding supports our proposal that the expanded hydrophobic pocket provides a substrate-binding site for guaiacol. The reconstituted protein was found to accelerate the H2O2dependent oxidation of small substrates, such as guaiacol, 2,2′C

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of metal complexes are strongly influenced by the structure of the ligand framework.

function of myoglobin is to store O2, appropriate modification of myoglobin can convert it to a useful peroxidase enzyme.

Peroxidation

Table 1. Kinetic Parameters for Guaiacol Oxidation Catalyzed by Modified Myoglobins at 25 °C, pH 6.0 myoglobin a

nMb MbH64Da rMb(2)a rMbH64D(2)a rMbH64D(3)b nHRPc

Km (mM)

kcat (s−1)

kcat/Km (M−1·s−1)

54 ± 15 1.8 ± 0.4 3.4 ± 0.6 0.052 ± 0.016 0.29 ± 0.06 5.8 ± 0.7

2.8 ± 0.6 9.0 ± 1.2 6.2 ± 0.6 1.2 ± 0.1 24 ± 2 420 ± 40

53 5100 1800 23000 85000 72000

Iron porphycene 5 was first prepared as an artificial cofactor of myoglobin to regulate O2 affinity and O2/CO discrimination and the obtained reconstituted myoglobin, rMb(5), was well characterized by EPR spectroscopy and X-ray crystal structure analysis with 2.25 Å resolution.17,18 Further, it was found that rMb(5) has peroxidase activity toward guaiacol, thioanisole, and styrene oxidation under neutral pH conditions. Compared to nMb, rMb(5) accelerates the H2O2-dependent oxidation of guaiacol by 11-fold at pH 7.0, indicating that 5 is a suitable cofactor to provide enhancement of peroxidase activity in the protein scaffold.18 Next, we focused on incorporating 5 into apoHRP after removal of heme from nHRP and then obtained the reconstituted protein rHRP(5). The initial turnover number of rHRP(5) for thioanisole oxidation was found to be 10-fold greater than the turnover number of nHRP, suggesting that the peroxidase activity of rHRP(5) clearly exceeds that of a native peroxidase enzyme.19 An additional important result of this work was that a compound I-like intermediate, which is formally oxidized by two electron equivalents above the resting ferric species 5, was detected by stopped-flow analysis. Characteristic and transient absorption arising from a porphycene π-cation species (Pc+•) was monitored at 700−900 nm over 40 ms following addition of H2O2 (Figure 5). The kinetic study

a

Reference 13. bReference 14. cReference 15.

Deformylation

Cytochrome P450 (P450) enzymes require flavoprotein as an electron donor and accept a total of two electrons from an electron mediator via P450−flavoprotein complexation during catalysis of oxygen transfer reactions. To engineer myoglobin to provide an example of P450 activity, we prepared flavoheme 4 where the isoalloxazine moiety of flavin is attached to the terminus of one of the heme-propionate side chains and inserted 4 into apomyoglobin to yield flavomyoglobin, rMb(4). The addition of NADH to a solution of rMb(4) did not provide any hydroxylation products from alkane substrates in this system. In contrast, deformylation of 2-phenylpropionaldehyde, which is another enzymatic P450 reaction, was observed and gave acetophenone as a product with an initial turnover frequency (TOF) of 0.77 s−1 in the presence of superoxide dismutase (SOD) and catalase (Scheme 1).16 This result suggests that the Scheme 1. Deformylation Catalyzed by Flavomyoglobin rMb(4)

flavin moiety located near the heme pocket works as an electron mediator and the peroxoanion intermediate (Fe(III)−O22−) could be produced via heme reduction and dioxygen binding.

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NON-NATURAL METALLOPORPHYRINOID COFACTORS

Porphyrinoid Framework

In nature, porphyrin functions as a ligand with highly symmetrical dianionic character. Iron in the porphyrin framework is stabilized as ferrous, ferric, and ferryl species within protein scaffolds. Corrin and corphin are non-porphyrin macrocyclic ligands naturally found in cobalamin and F430 pigments, respectively. Several artificial porphyrinoids have been synthesized and are known to possess clearly different characteristics relative to the porphyrin ligand. In investigating a series of porphyrinoids, our group has particularly focused on porphycene, corrole and tetradehydrocorrin as artificial cofactors for hemoproteins. Porphycene is a constitutional isomer of porphyrin with D2h symmetry. Corrole and corrin lack one meso carbon compared to the porphyrin framework, providing a trianionic ligand and a monoanionic ligand, respectively. The physicochemical properties and reactivities

Figure 5. Transient absorption spectra of rHRP(5) over 40 ms upon addition of H2O2 at 10 °C, pH 7.0.

indicates that the reactivity of the intermediate, Fe(IV)OPc+•, is significantly higher (ca. more than 100-fold) than that of native heme 1 in the HRP scaffold, although the rate of the intermediate formation of 5 is almost same as that of 1. Corrole is one of the most highly investigated porphyrinoids in chemistry and metallocorroles have been tested as oxidation catalysts. In particular, the corrole ligand is expected to be capable of stabilizing high-valent metal species. We have prepared iron corrole 6 as an artificial cofactor for apomyoglobin and apoHRP and obtained both reconstituted proteins, rMb(6) D

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Accounts of Chemical Research and rHRP(6), respectively.20 Based on 1H NMR measurements, the resting state of 6 in the myoglobin scaffold is the ferric state with S = 3/2, whereas the HRP scaffold will stabilize the ferryl state or lead to the ferric state with a corrole π-cation radical. As a result, it is found that rMb(6) remarkably accelerates H2O2dependent guaiacol oxidation, whereas the peroxidase activity of rHRP(6) is decreased relative to native HRP (Figure 6).

Figure 6. Time course of guaiacol oxidation catalyzed by Mbs and HRPs upon addition of excess amounts of H2O2 at 20 °C, pH 7.0.

Cyclopropanation

Cyclopropanation of olefin molecules by ethyl diazoacetate (EDA) is one of the most interesting abiological reactions catalyzed by artificial metalloenzymes.21,22 Hemoproteins and these mutants where Fe in protoporphyrin IX is replaced with Ir, Rh, Ru, etc., show high catalytic activities for the reactions.23−27 The reactive carbene intermediate species has not been spectroscopically detected in the protein-based catalysts, despite recent efforts to do so using crystal structures.28,29 Our group has found that rMb(5) is an efficient catalyst for cyclopropanation via the spectroscopically detectable active metallocarbene species (Figure 7a).30 The overall catalysis for cyclopropanation of styrene with EDA is also accelerated by 26-fold by rMb(5) relative to nMb (Figure 7b). The reactions of rMb(5) and nMb with EDA were further evaluated by observing transient absorption spectral changes using stopped flow techniques. From the kinetics of the absorption changes, the second-order rate constants, k, for rMb(5) and nMb were determined to be 2.5 × 10−1 and 4.0 × 10−4 mM−1·s−1, respectively. Furthermore, the detectable intermediate species was converted to the resting state of ferrous rMb(5) upon addition of styrene. In the DFT calculations to investigate the reactions, a smaller kinetic barrier was theoretically produced for rMb(5) relative to nMb. This difference is likely due to the stronger ligand field effect of porphycene providing stabilization of the triplet of resting state compared with porphyrin. These findings contribute to the rational design of highly active artificial metalloenzymes that can catalyze abiological reactions via the carbene intermediate.

Figure 7. (a) Catalytic cycle of styrene cyclopropanation via the active carbene intermediate in rMb(5). (b) Plots of TOF for styrene cyclopropanation using EDA at pH 8.0, 25 °C. [Mb] = 4.3 μM; [styrene] = 2.0 mM.

enzymes. In contrast, myoglobin reconstituted with manganese porphycene 7 (rMb(7)) was found to be capable of H2O2dependent catalytic hydroxylation of inert C(sp3)−H bonds with a spectroscopically detectable active species.31,32 The 1:1 binding of 7 to apomyoglobin is supported by two crystal structures of rMb(7) using crystals obtained at pH 7.0 and 8.5 (Figure 8). In this structure, the manganese center is coordinated by His93, the intrinsic axial ligand to heme in nMb, and a water molecule as the sixth ligand. The catalytic hydroxylation of the C(sp3)−H bond of ethylbenzene was evaluated using H2O2 as a terminal oxidant (Scheme 2). It was found that rMb(7) produces 1-phenylethanol as a product with a turnover number of 13 without any byproducts, whereas nMb, rMb(5), and Mn-substituted Mb do not generate any products under the same conditions. Interestingly, 7 itself does not catalyze this reaction, indicating that the protein matrix is essential for the formation of the active species or enhancement of the hydroxylation reactivity. Further work to carry out the hydroxylation of small alkanes is now in progress.

Hydroxylation

Myoglobin does not have enzymatic activity toward alkane hydroxylation, despite having the same cofactor as P450 E

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Figure 9. Crystal structures of rMb(8) before and after reduction.

although it is limited to an intraprotein reaction. Similarly, a nickel tetradehydrocorrin derivative in a protein matrix is under investigation to replicate the function of the F430-dependent enzyme.

Figure 8. Crystal structure of rMb(7) at pH 8.5.

Scheme 2. Hydroxylation of Ethylbenzene Catalyzed by rMb(7)

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ORGANOMETALLIC CATALYSTS COFACTOR

Hydrogenase-like Reaction

Hydrogenase (H2ase) catalyzes the reversible interconversion of H+ and H2 under mild conditions. One of the common H2ases, [FeFe]-H2ase, has a diiron core with a characteristic bridging azadithiolate ligand and diatomic ligands such as CO and CN− in the active site (H-cluster).40 To replicate the H-cluster, several groups have prepared molecular catalysts with a single or multiple component visible light-driven photochemical systems, which provide electrons into a cascade for the catalysis.41 Furthermore, to produce a water-soluble model, the dithiolate bridging ligands of the H-cluster were mimicked by a designed peptide, where two cysteines located on the same face of the αhelix at the i and the i + 3 positions are provided to replicate the functions of the dithiolate ligand.42 Our group has applied a similar dative anchoring approach to incorporate a photocatalytically active diiron core into a Cys-X-Y-Cys fragment of apo-cytochrome c (apo-Cyt c).43,44 After the removal of heme c in Cyt c, diiron nonacarbonyl (Fe2(CO)9) was incorporated into apo-Cyt c to form a [Fe2(μ-S(Cys))(CO)6] core 9 in the protein scaffold (Figure 10). The obtained conjugate, rCyt c(9), was

The mechanism of the hydroxylation catalyzed by rMb(7) has also been investigated. Kinetic isotope effects for the catalytic reaction indicate that C(sp3)−H bond activation is one of the rate-limiting steps and 18O-labeling experiments using H218O2 indicate that the reaction follows an OH-rebound process. These results support the speculation that the reaction mechanism is similar to that proposed for P450 enzymes.33 Interestingly, the active species was successfully monitored by spectroscopic methods. The half-life of the active species is 40 s, and no near-infrared (NIR) absorption bands were observed. No signals in EPR spectra were observed with perpendicular and parallel modes. The spectroscopic data obtained for rMb(7) provide evidence for the formation of the Mn(V)oxo species as the compound I-like active intermediate, whereas the Mn(IV)oxo species with a porphycene π-cation radical is ruled out. It was concluded that the Mn(V)oxo species has only moderate reactivity, which will not oxidize the protein matrix and promote the C−H bond hydroxylation of external substrates in the heme pocket via a mechanism similar to that of the P450s. Methyl Transfer Reaction

In most mammals and bacteria, cobalamin-dependent methionine synthase catalyzes an essential methylation of homocysteine to form methionine. The enzyme has a large protein matrix and a methylcobalamin cofactor. The large size of the enzyme causes challenges in elucidation of its reaction mechanisms.34 In this context, a cobalt tetradehydrocorrin derivative 8 was designed as a model of cobalamin, and rMb(8) was prepared and investigated as a hybrid model to replicate the reaction promoted by methionine synthase.35−39 The Co(II) species of rMb(8) was first prepared, and its crystal structure revealed ligation of His93 to the Co center in the heme pocket (Figure 9). Soaking the Co(II) crystals in a dithionite solution successfully provided the crystals to the Co(I) species. Interestingly, the tetracoordinated Co(I) species formed by axial histidine flipping was directly observed in the crystal structure. In the heme pocket, the Co(I) species further reacts with methyl iodide to form the CH3−Co(III) species, which promotes subsequent transmethylation to the Nε2 atom of the His64 imidazole ring in rMb(8) at 25 °C over a period of 24 h. These findings indicate that the methyl group transfer reaction is promoted via the methylated cobalt complex as seen in methionine synthase,

Figure 10. Plausible structure of the reaction site in rCyt c(9) containing a diiron hexacarbonyl cluster, which catalyzes photodriven H2 evolution.

found to produce H2 in the presence of [Ru(bpy)3]2+ as a photosensitizer and ascorbate as a sacrificial reagent. Photocatalytic production of H2 reached a plateau after 2 h, yielding a turnover number (TON) of ∼80 per rCyt c(9) with a maximum TOF of ∼2.1 min−1. In contrast, a small model where a diiron cluster is formed with a heptapeptide fragment YKCAQCH was found to promote H2 production with a TOF of 0.47 min−1. This indicates that the hemoprotein environment near the diiron core provides a suitable platform for efficient photocatalytic H2 production. Next, we focused on nitrobindin (NB), an NO-binding hemoprotein, as a protein scaffold, because apoNB has a rigid βF

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Scheme 3. Representative Reaction Schemes of (a) RCM and (b) ROMP

Figure 11. Crystal structures of (a) a holo-form and (b) an apo-form of nitrobindin (PDB 3EMM and 3WJB).

rNB4(12) was found to have ROMP activity with a TON of 1100 under 0.008 mol % catalyst loading. Furthermore, rNB11(12) has remarkably high activity with a TON of 9700 relative to rNB11(11).

cysteine residues, a mutant M75L/Q96C/M148L was produced and an [Fe2(μ-S)2(CO)6] core with a tethered maleimide moiety, 10, was incorporated into the cavity of the mutant apoprotein to form a covalent linkage between the maleimide moiety and a cysteine residue in the β-barrel cavity (Figure 12).45 This conjugate is designated as rNBM75L/Q96C/M148L(10).

Phenylacetylene Polymerization

Our group has also reported that a covalently anchored Rh(I) complex 13 with cyclopentadiene (Cp) and cyclooctadiene ligands promotes the polymerization of phenylacetylene (PA) within the β-barrel cavity of NB. The Rh(I) complex was linked to a specifically engineered Cys residue at the rim of apoNB via a maleimide group. The PA polymerization catalyzed by the hybrid protein rNBM75L/Q96C/M148L(13) was found to proceed smoothly, giving the polymer (PPA) with an Mn value of 42 600 at 25 °C, pH 8 (Scheme 4). The trans/cis ratio of PPA was found Scheme 4. Phenylacetylene Polymerization Catalyzed by a Rhodium Complex-Linked Hybrid

Figure 12. An MD structure of rNBM75L/Q96C/M148L(10).

Photochemical H2 production experiments for this conjugate were performed in the presence of [Ru(bpy)3]2+ and excess amounts of ascorbate. The photocatalytic production of H2 reached a plateau after 6 h, yielding a TON of ∼130 per hybrid catalyst with a maximum TOF of ∼2.3 min−1. Olefin Metathesis

to be 54/46, whereas the Rh-complex alone predominantly provided PPA with a trans/cis ratio of 7/93. These results indicate that the NB cavity regulates the approach of the monomer, leading to the generation of trans-PPA.51 Reengineering of the NB cavity by computationally guided site-directed mutagenesis significantly improves the trans/cis stereoselectivity in phenylacetylene polymerization. Particularly, the hybrid catalysts rNB4(13) and rNB5(13) (NB5, F44W/M75L/H76L/ Q96C/M148L/H158L NB mutant) mainly provide trans-PPA (up to 82/18 trans/cis ratio for rNB4(13)), indicating that the stereoselectivity depends on the structure of the protein scaffold near the Rh complex. X-ray crystal structure and MD simulation results give insights into how stereopreference is guided within the hybrid catalysts and how monomer access to the rhodium center is controlled (Figure 13).52

Several groups have recently focused on olefin metathesis and investigated incorporation of the Hoveyda−Grubbs (HG) type ruthenium carbene complex into a protein scaffold.46−49 In a collaboration between Okuda’s group and our group, the HG catalysts 11 and 12 were covalently anchored within the cavity of two nitrobindin mutants, which mainly differ in the size of the cavity volume. The so-called NB4 mutant (NB4, M75L/H76L/ Q96C/M148L/H158L NB mutant) has a cavity with a volume of 855 Å3, whereas the NB11 mutant (NB11, M75L/H76L/ Q96C/M148L/H158A NB mutant) has an increased cavity volume of 1161 Å3. These biohybrid catalysts, rNB11(11), rNB4(12), and rNB11(12), were found to catalyze ring-closing metathesis (RCM) and ring-opening metathesis polymerization (ROMP) of water-soluble substrates.50 Particularly, rNB11(12) exhibits remarkably high RCM activity for 4,4-bis(hydroxymethyl)-1,6-heptadiene (Scheme 3). With decreased catalyst loading of 0.33 mol % under mild conditions (pH = 6.0, at 40 °C) the TON increases to 178. Next, the ROMP reaction was performed with the 7-oxanorbornene derivative. The protein-free HG-type catalyst showed no activity in aqueous buffer solutions under only 0.01 mol % catalyst loading, whereas

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CONCLUSION The heme pockets in a series of hemoproteins usually support inherent physicochemical events by providing a hemecoordination sphere. In contrast, after removal of native heme, the vacant heme pockets provide us with a useful cavity for G

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Koji Oohora: 0000-0003-1155-6824 Akira Onoda: 0000-0002-5791-4386 Takashi Hayashi: 0000-0002-2215-935X Notes

The authors declare no competing financial interest. Biographies Koji Oohora received his Ph.D. degree from Osaka University in 2011. He has been an Assistant Professor at Osaka University since 2011. His research interests involve creation of metalloprotein-based catalysts and materials. Akira Onoda received his Ph.D. degree from Osaka University in 2002. He has been an Associate Professor of Applied Chemistry at Osaka University since 2008. His research focuses on the interface of bioinorganic chemistry, biohybrid catalysts, biomaterials, and bionanotechnology. Takashi Hayashi received his Ph.D. degree from Kyoto University in 1991. After accepting an Assistant Professor position at Kyoto University in 1990, he conducted research at the Scripps Research Institute as a visiting scientist from 1995 to 1996. He moved to Kyushu University as an Associate Professor in 1997 and was then promoted to a full Professor at Osaka University in 2005. His current research interests are in bioinorganic chemistry, supramolecular chemistry, and porphyrin chemistry.

Figure 13. (a) Crystal structure of rNB4(13) covalently linking a CpRh complex and (b) close-up view of the CpRh complex within the cavity. The replaced residues are shown in stick representation.

development of artificial catalytic scaffolds. Here, we have described several biohybrid catalysts that are produced by combining a reactive metal complex and a suitable protein matrix originally derived from relatively simple hemoproteins such as myoglobin, horseradish peroxidase, cytochrome c, and nitrobindin. The topics described in this Account can be mainly grouped into the following two categories: (1) reconstitution of hemoprotein with modified metalloporphyrins or non-natural metalloporphyrinoids as cofactors and (2) construction of bioorganometallic catalysts by incorporation of organometallic species into protein scaffolds. These strategies enable us to obtain new biocatalysts which have remarkably enhanced activity, the ability to control stereo- and regioselectivity, and the ability to promote reactions different from those seen in nature. These attractive results are mainly derived from the incorporation of various metal complexes that are not included in nature’s tool box. Until now, many biochemists have improved enzymes by sitedirected mutagenesis, laboratory evolution,53 or chemical modifications of amino acid residues in the protein. In contrast, the more recently developed methods described in this Account will dramatically expand opportunities to create various examples of new metal-dependent artificial enzymes, because many metal complexes are known to chemists and various heme pockets are now available for use as scaffolds to support the metal complexes. Looking to the future, we believe that appropriate combinations of non-natural metal complexes and appropriately modified protein cavities will have significant potential for development of new metalloproteins capable of promoting reactions different from those seen in nature or difficult reactions where controlling selectivity or reactivity in conventional organic synthesis is required.

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ACKNOWLEDGMENTS The graduate students and postdoctoral associate who contributed to the results presented in this article are gratefully acknowledged, as well as our collaborators named in the references cited. We gratefully acknowledge support from Grants-in-Aid for Scientific Research provided by JSPS KAKENHI Grant Numbers JP15H05804, JP15KT0144, JP16K14036, JP16H06045, JP16H00837, JP17H05370, JP18K19099, JP18KK0156, JP18H04651, and JST PRESTO (JPMJPR15S2).

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