Hartwig, J.F.

Nature 2016, 534, 534-537, 10.1038/nature17968

Enzymes that contain metal ions—that is, metalloenzymes—possess the reactivity of a transition metal centre and the potential of molecular evolution to modulate the reactivity and substrate-selectivity of the system1. By exploiting substrate promiscuity and protein engineering, the scope of reactions catalysed by native metalloenzymes has been expanded recently to include abiological transformations2,3. However, this strategy is limited by the inherent reactivity of metal centres in native metalloenzymes. To overcome this limitation, artificial metalloproteins have been created by incorporating complete, noble-metal complexes within proteins lacking native metal sites1,4,5. The interactions of the substrate with the protein in these systems are, however, distinct from those with the native protein because the metal complex occupies the substrate binding site. At the intersection of these approaches lies a third strategy, in which the native metal of a metalloenzyme is replaced with an abiological metal with reactivity different from that of the metal in a native protein6,7,8. This strategy could create artificial enzymes for abiological catalysis within the natural substrate binding site of an enzyme that can be subjected to directed evolution. Here we report the formal replacement of iron in Fe-porphyrin IX (Fe-PIX) proteins with abiological, noble metals to create enzymes that catalyse reactions not catalysed by native Fe-enzymes or other metalloenzymes9,10. In particular, we prepared modified myoglobins containing an Ir(Me) site that catalyse the functionalization of C–H bonds to form C–C bonds by carbene insertion and add carbenes to both β-substituted vinylarenes and unactivated aliphatic α-olefins. We conducted directed evolution of the Ir(Me)-myoglobin and generated mutants that form either enantiomer of the products of C–H insertion and catalyse the enantio- and diastereoselective cyclopropanation of unactivated olefins. The presented method of preparing artificial haem proteins containing abiological metal porphyrins sets the stage for the generation of artificial enzymes from innumerable combinations of PIX-protein scaffolds and unnatural metal cofactors to catalyse a wide range of abiological transformations.

Niemeyer, C.M.

Chem. - Asian J. 2009, 4, 1064-1069, 10.1002/asia.200900082

A hybrid myoglobin, containing a single‐stranded DNA anchor and a redox‐active ruthenium moiety tethered to the heme center can be used as a photocatalyst. The catalyst can be selectively immobilized on a surface‐bound complementary DNA molecule and thus readily recycled from complex reaction mixtures. This principle may be applied to a range of heme‐dependent enzymes allowing the generation of novel light‐triggered photocatalysts. Photoactivatable myoglobin containing a DNA oligonucleotide as a structural anchor was designed by using the reconstitution of artificial heme moieties containing Ru3+ ions. This semisynthetic DNA–enzyme conjugate was successfully used for the oxidation of peroxidase substrates by using visible light instead of H2O2 for the activation. The DNA anchor was utilized for the immobilization of the enzyme on the surface of magnetic microbeads. Enzyme activity measurements not only indicated undisturbed biofunctionality of the tethered DNA but also enabled magnetic separation‐based enrichment and recycling of the photoactivatable biocatalyst.

Lu, Y.

Angew. Chem. Int. Ed. 2012, 51, 5589-5592, 10.1002/anie.201201981

Rational design of functional enzymes with a high number of turnovers is a challenge, especially those with a complex active site, such as respiratory oxidases. Introducing two His and one Tyr residues into myoglobin resulted in enzymes that reduce O2 to H2O with more than 1000 turnovers (red line, see scheme) and minimal release of reactive oxygen species. The positioning of the Tyr residue is critical for activity.

Lu, Y.; Wang, J.

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

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

Green, A.P.; Hilvert, D.

J. Am. Chem. Soc. 2018, 140, 1535-1543, 10.1021/jacs.7b12621

Expanding the range of genetically encoded metal coordination environments accessible within tunable protein scaffolds presents excellent opportunities for the creation of metalloenzymes with augmented properties and novel activities. Here, we demonstrate that installation of a noncanonical Nδ-methyl histidine (NMH) as the proximal heme ligand in the oxygen binding protein myoglobin (Mb) leads to substantial increases in heme redox potential and promiscuous peroxidase activity. Structural characterization of this catalytically modified myoglobin variant (Mb NMH) revealed significant changes in the proximal pocket, including alterations to hydrogen-bonding interactions involving the prosthetic porphyrin cofactor. Further optimization of Mb NMH via a combination of rational modification and several rounds of laboratory evolution afforded efficient peroxidase biocatalysts within a globin fold, with activities comparable to those displayed by nature’s peroxidases.

Lu, Y.

J. Am. Chem. Soc. 2004, 126, 10812-10813, 10.1021/ja046908x

Introducing nonnative metal ions or metal-containing prosthetic groups into a protein can dramatically expand the repertoire of its functionalities and thus its range of applications. Particularly challenging is the control of substrate-binding and thus reaction selectivity such as enantioselectivity. To meet this challenge, both non-covalent and single-point attachments of metal complexes have been demonstrated previously. Since the protein template did not evolve to bind artificial metal complexes tightly in a single conformation, efforts to restrict conformational freedom by modifying the metal complexes and/or the protein are required to achieve high enantioselectivity using the above two strategies. Here we report a novel site-selective dual anchoring (two-point covalent attachment) strategy to introduce an achiral manganese salen complex (Mn(salen)), into apo sperm whale myoglobin (Mb) with bioconjugation yield close to 100%. The enantioselective excess increases from 0.3% for non-covalent, to 12.3% for single point, and to 51.3% for dual anchoring attachments. The dual anchoring method has the advantage of restricting the conformational freedom of the metal complex in the protein and can be generally applied to protein incorporation of other metal complexes with minimal structural modification to either the metal complex or the protein.

Fruk, L.; Niemeyer, C.M.

Croat. Chem. Acta 2011, 84, 269-275, 10.5562/cca1828

A bimetallic heme-DNA cofactor, containing an iron and a copper center, was synthesized for the design of novel hybrid catalysts for stereoselective synthesis. The cofactor was used for the reconstitution of apo-myoglobin. Both the cofactor alone and its myoglobin adduct were used to catalyze a model Diels Alder reaction. Stereoselectivity of this conversion was analyzed by chiral HPLC. Reactions carried out in the presence of myoglobin-heme-Cu-DNA catalyst showed greater product conversion and stereoselectivity than those carried out with the heme-Cu-DNA cofactor. This observation suggested that the protein shell plays a significant role in the catalytic conversion.

Hilvert, D.

Nat. Catal. 2018, 1, 578-584, 10.1038/s41929-018-0105-6

Non-canonical amino acid ligands are useful for fine-tuning the catalytic properties of metalloenzymes. Here, we show that recombinant replacement of the histidine ligand proximal to haem in myoglobin with Nδ-methylhistidine enhances the protein’s promiscuous carbene-transfer chemistry, enabling efficient styrene cyclopropanation in the absence of reductant, even under aerobic conditions. The increased electrophilicity of the modified Fe(iii) centre, combined with subtle structural adjustments at the active site, allows direct attack of ethyl diazoacetate to produce a reactive carbenoid adduct, which has an unusual bridging Fe(iii)–C–N(pyrrole) configuration as shown by X-ray crystallography. Quantum chemical calculations suggest that the bridged complex equilibrates with the more reactive end-on isomer, ensuring efficient cyclopropanation. These findings underscore the potential of non-canonical ligands for extending the capabilities of metalloenzymes by opening up new reaction pathways and facilitating the characterization of reactive species that would not otherwise accumulate.

Hasegawa, J.-Y.; Lehnert, N.

J. Am. Chem. Soc. 2017, 139, 17265-17268, 10.1021/jacs.7b10154

Myoglobin reconstituted with iron porphycene catalyzes the cyclopropanation of styrene with ethyl diazoacetate. Compared to native myoglobin, the reconstituted protein significantly accelerates the catalytic reaction and the kcat/Km value is 26-fold enhanced. Mechanistic studies indicate that the reaction of the reconstituted protein with ethyl diazoacetate is 615-fold faster than that of native myoglobin. The metallocarbene species reacts with styrene with the apparent second-order kinetic constant of 28 mM–1 s–1 at 25 °C. Complementary theoretical studies support efficient carbene formation by the reconstituted protein that results from the strong ligand field of the porphycene and fewer intersystem crossing steps relative to the native protein. From these findings, the substitution of the cofactor with an appropriate metal complex serves as an effective way to generate a new biocatalyst.

Lu, Y.

J. Am. Chem. Soc. 2006, 128, 6766-6767, 10.1021/ja058822p

The effects of metal ions on the reduction of nitric oxide (NO) with a designed heme copper center in myoglobin (F43H/L29H sperm whale Mb, CuBMb) were investigated under reducing anaerobic conditions using UV−vis and EPR spectroscopic techniques as well as GC/MS. In the presence of Cu(I), catalytic reduction of NO to N2O by CuBMb was observed with turnover number of 2 mol NO·mol CuBMb-1·min-1, close to 3 mol NO·mol enzyme-1·min-1 reported for the ba3 oxidases from T. thermophilus. Formation of a His-heme-NO species was detected by UV−vis and EPR spectroscopy. In comparison to the EPR spectra of ferrous-CuBMb-NO in the absence of metal ions, the EPR spectra of ferrous-CuBMb-NO in the presence of Cu(I) showed less-resolved hyperfine splitting from the proximal histidine, probably due to weakening of the proximal His-heme bond. In the presence of Zn(II), formation of a five-coordinate ferrous-CuBMb-NO species, resulting from cleavage of the proximal heme Fe-His bond, was shown by UV−vis and EPR spectroscopic studies. The reduction of NO to N2O was not observed in the presence of Zn(II). Control experiments using wild-type myoglobin indicated no reduction of NO in the presence of either Cu(I) or Zn(II). These results suggest that both the identity and the oxidation state of the metal ion in the CuB center are important for NO reduction. A redox-active metal ion is required to deliver electrons, and a higher oxidation state is preferred to weaken the heme iron−proximal histidine toward a five-coordinate key intermediate in NO reduction.

Artero, V.

Inorg. Chem. 2014, 53, 8071-8082, 10.1021/ic501014c

Cobaloximes are popular H2 evolution molecular catalysts but have so far mainly been studied in nonaqueous conditions. We show here that they are also valuable for the design of artificial hydrogenases for application in neutral aqueous solutions and report on the preparation of two well-defined biohybrid species via the binding of two cobaloxime moieties, {Co(dmgH)2} and {Co(dmgBF2)2} (dmgH2 = dimethylglyoxime), to apo Sperm-whale myoglobin (SwMb). All spectroscopic data confirm that the cobaloxime moieties are inserted within the binding pocket of the SwMb protein and are coordinated to a histidine residue in the axial position of the cobalt complex, resulting in thermodynamically stable complexes. Quantum chemical/molecular mechanical docking calculations indicated a coordination preference for His93 over the other histidine residue (His64) present in the vicinity. Interestingly, the redox activity of the cobalt centers is retained in both biohybrids, which provides them with the catalytic activity for H2 evolution in near-neutral aqueous conditions.

Watanabe, Y.

J. Am. Chem. Soc. 2005, 127, 6556-6562, 10.1021/ja045995q

New methods for the synthesis of artificial metalloenzymes are important for the construction of novel biocatalysts and biomaterials. Recently, we reported new methodology for the synthesis of artificial metalloenzymes by reconstituting apo-myoglobin with metal complexes (Ohashi, M. et al., Angew Chem., Int. Ed.2003, 42, 1005−1008). However, it has been difficult to improve their reactivity, since their crystal structures were not available. In this article, we report the crystal structures of MIII(Schiff base)·apo-A71GMbs (M = Cr and Mn). The structures suggest that the position of the metal complex in apo-Mb is regulated by (i) noncovalent interaction between the ligand and surrounding peptides and (ii) the ligation of the metal ion to proximal histidine (His93). In addition, it is proposed that specific interactions of Ile107 with 3- and 3‘-substituent groups on the salen ligand control the location of the Schiff base ligand in the active site. On the basis of these results, we have successfully controlled the enantioselectivity in the sulfoxidation of thioanisole by changing the size of substituents at the 3 and 3‘ positions. This is the first example of an enantioselective enzymatic reaction regulated by the design of metal complex in the protein active site.

Hayashi, T

Inorg. Chem. 2006, 45, 10530-10536, 10.1021/ic061130x

The incorporation of an artificially created metal complex into an apomyoglobin is one of the attractive methods in a series of hemoprotein modifications. Single crystals of sperm whale myoglobin reconstituted with 13,16-dicarboxyethyl-2,7-diethyl-3,6,12,17-tetramethylporphycenatoiron(III) were obtained in the imidazole buffer, and the 3D structure with a 2.25-Å resolution indicates that the iron porphycene, a structural isomer of hemin, is located in the normal position of the heme pocket. Furthermore, it was found that the reconstituted myoglobin catalyzed the H2O2-dependent oxidations of substrates such as guaiacol, thioanisole, and styrene. At pH 7.0 and 20 °C, the initial rate of the guaiacol oxidation is 11-fold faster than that observed for the native myoglobin. Moreover, the stopped-flow analysis of the reaction of the reconstituted protein with H2O2 suggested the formation of two reaction intermediates, compounds II- and III-like species, in the absence of a substrate. It is a rare example that compound III is formed via compound II in myoglobin chemistry. The enhancement of the peroxidase activity and the formation of the stable compound III in myoglobin with iron porphycene mainly arise from the strong coordination of the Fe−His93 bond.

Hayashi, T

J. Am. Chem. Soc. 2013, 135, 17282-17285, 10.1021/ja409404k

Myoglobin reconstituted with manganese porphycene was prepared in an effort to generate a new biocatalyst and was characterized by spectroscopic techniques. The X-ray crystal structure of the reconstituted protein reveals that the artificial cofactor is located in the intrinsic heme-binding site with weak ligation by His93. Interestingly, the reconstituted protein catalyzes the H2O2-dependent hydroxylation of ethylbenzene to yield 1-phenylethanol as a single product with a turnover number of 13 at 25 °C and pH 8.5. Native myoglobin and other modified myoglobins do not catalyze C–H hydroxylation of alkanes. Isotope effect experiments yield KIE values of 2.4 and 6.1 for ethylbenzene and toluene, respectively. Kinetic data, log kobs versus BDE(C(sp3)–H) for ethylbenzene, toluene, and cyclohexane, indicate a linear relationship with a negative slope. These findings clearly indicate that the reaction occurs via a rate-determining step that involves hydrogen-atom abstraction by a Mn(O) species and a subsequent rebound hydroxylation process which is similar to the reaction mechanism of cytochrome P450.

Lu, Y.; Wang, J.

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

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

Fasan, R.; Khare, S.D.

Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 12472-12477, 10.1073/pnas.1708907114

Thermostabilization represents a critical and often obligatory step toward enhancing the robustness of enzymes for organic synthesis and other applications. While directed evolution methods have provided valuable tools for this purpose, these protocols are laborious and time-consuming and typically require the accumulation of several mutations, potentially at the expense of catalytic function. Here, we report a minimally invasive strategy for enzyme stabilization that relies on the installation of genetically encoded, nonreducible covalent staples in a target protein scaffold using computational design. This methodology enables the rapid development of myoglobin-based cyclopropanation biocatalysts featuring dramatically enhanced thermostability (ΔTm = +18.0 °C and ΔT50 = +16.0 °C) as well as increased stability against chemical denaturation [ΔCm (GndHCl) = 0.53 M], without altering their catalytic efficiency and stereoselectivity properties. In addition, the stabilized variants offer superior performance and selectivity compared with the parent enzyme in the presence of a high concentration of organic cosolvents, enabling the more efficient cyclopropanation of a water-insoluble substrate. This work introduces and validates an approach for protein stabilization which should be applicable to a variety of other proteins and enzymes.

Watanabe, Y.

J. Am. Chem. Soc. 2004, 126, 436-437, 10.1021/ja038798k

To modulate the physiological function of a hemoprotein, most approaches have been demonstrated by site-directed mutagenesis. Replacement of the native heme with an artificial prosthetic group is another way to modify a hemoprotein. However, an alternate method, mutation or heme reconstitution, does not always demonstrate sufficient improvement compared with the native heme enzyme. In the present study, to convert a simple oxygen storage hemoprotein, myoglobin, into an active peroxidase, we applied both methods at the same time. The native heme of myoglobin was replaced with a chemically modified heme 2 having two aromatic rings at the heme-propionate termini. The constructed myoglobins were examined for 2-methoxyphenol (guaiacol) oxidation in the presence of H2O2. Compared with native myoglobin, rMb(H64D·2) showed a 430-fold higher kcat/Km value, which is significantly higher than that of cytochrome c peroxidase and only 3-fold less than that of horseradish peroxidase. In addition, myoglobin-catalyzed degradation of bisphenol A was examined by HPLC analysis. The rMb(H64D·2) showed drastic acceleration (>35-fold) of bisphenol A degradation compared with the native myoglobin. In this system, a highly oxidized heme reactive species is smoothly generated and a substrate is effectively bound in the heme pocket, while native myoglobin only reversibly binds dioxygen. The present results indicate that the combination of a modified-heme reconstitution and an amino acid mutation should offer interesting perspectives toward developing a useful biomolecule catalyst from a hemoprotein.

Fasan, R.

Bioorg. Med. Chem. 2014, 22, 5697-5704, 10.1016/j.bmc.2014.05.015

The direct conversion of aliphatic CH bonds into CN bonds provides an attractive approach to the introduction of nitrogen-containing functionalities in organic molecules. Following the recent discovery that cytochrome P450 enzymes can catalyze the cyclization of arylsulfonyl azide compounds via an intramolecular C(sp3)H amination reaction, we have explored here the CH amination reactivity of other hemoproteins. Various heme-containing proteins, and in particular myoglobin and horseradish peroxidase, were found to be capable of catalyzing this transformation. Based on this finding, a series of engineered and artificial myoglobin variants containing active site mutations and non-native Mn- and Co-protoporphyrin IX cofactors, respectively, were prepared to investigate the effect of these structural changes on the catalytic activity and selectivity of these catalysts. Our studies showed that metallo-substituted myoglobins constitute viable CH amination catalysts, revealing a distinctive reactivity trend as compared to synthetic metalloporphyrin counterparts. On the other hand, amino acid substitutions at the level of the heme pocket were found to be beneficial toward improving the stereo- and enantioselectivity of these Mb-catalyzed reactions. Mechanistic studies involving kinetic isotope effect experiments indicate that CH bond cleavage is implicated in the rate-limiting step of myoglobin-catalyzed amination of arylsulfonyl azides. Altogether, these studies indicate that myoglobin constitutes a promising scaffold for the design and development of CH amination catalysts.

Lu, Y.

J. Am. Chem. Soc. 2010, 132, 9970-9972, 10.1021/ja103516n

A conserved 2-His-1-Glu metal center, as found in natural nonheme iron-containing enzymes, was engineered into sperm whale myoglobin by replacing Leu29 and Phe43 with Glu and His, respectively (swMb L29E, F43H, H64, called FeBMb(-His)). A high resolution (1.65 Å) crystal structure of Cu(II)-CN−-FeBMb(-His) was determined, demonstrating that the unique 2-His-1-Glu metal center was successfully created within swMb. The FeBMb(-His) can bind Cu, Fe, or Zn ions, with both Cu(I)-FeBMb(-His) and Fe(II)-FeBMb(-His) exhibiting nitric oxide reductase (NOR) activities. Cu dependent NOR activity was significantly higher than that of Fe in the same metal binding site. EPR studies showed that the reduction of NO to N2O catalyzed by these two enzymes resulted in different intermediates; a five-coordinate heme-NO species was observed for Cu(I)-FeBMb(-His) due to the cleavage of the proximal heme Fe-His bond, while Fe(II)-FeBMb(-His) remained six-coordinate. Therefore, both the metal ligand, Glu29, and the metal itself, Cu or Fe, play crucial roles in NOR activity. This study presents a novel protein model of NOR and provides insights into a newly discovered member of the NOR family, gNOR.

Oohora, K.

J. Am. Chem. Soc. 2017, 139, 18460-18463, 10.1021/jacs.7b11288

A mechanistic study of H2O2-dependent C–H bond hydroxylation by myoglobin reconstituted with a manganese porphycene was carried out. The X-ray crystal structure of the reconstituted protein obtained at 1.5 Å resolution reveals tight incorporation of the complex into the myoglobin matrix at pH 8.5, the optimized pH value for the highest turnover number of hydroxylation of ethylbenzene. The protein generates a spectroscopically detectable two-electron oxidative intermediate in a reaction with peracid, which has a half-life up to 38 s at 10 °C. Electron paramagnetic resonance spectra of the intermediate with perpendicular and parallel modes are silent, indicating formation of a low-spin MnV-oxo species. In addition, the MnV-oxo species is capable of promoting the hydroxylation of sodium 4-ethylbenzenesulfonate under single turnover conditions with an apparent second-order rate constant of 2.0 M–1 s–1 at 25 °C. Furthermore, the higher bond dissociation enthalpy of the substrate decreases the rate constant, in support of the proposal that the H-abstraction is one of the rate-limiting steps. The present engineered myoglobin serves as an artificial metalloenzyme for inert C–H bond activation via a high-valent metal species similar to the species employed by native monooxygenases such as cytochrome P450.

Hayashi, T

J. Am. Chem. Soc. 2009, 131, 15124-15125, 10.1021/ja907428e

Myoglobin (Mb) and horseradish peroxidase (HRP) were both reconstituted with a meso-unsubstituted iron corrole and their electronic configurations and peroxidase activities were investigated. The appearance of the 540 nm band upon incorporation of the iron corrole into apoMb indicates axial coordination by the proximal histidine imidazole in the Mb heme pocket. Based on 1H NMR measurements using the Evans method, the total magnetic susceptibility of the iron corrole reconstituted Mb was evaluated to be S = 3/2. In contrast, although a band does not appear in the vicinity of 540 nm during reconstitution of the iron corrole into the matrix of HRP, a spectrum similar to that of the iron corrole reconstituted Mb is observed upon the addition of dithionite. This observation suggests that the oxidation state of the corrole iron in the reconstituted HRP can be assigned as +4. The catalytic activities of both proteins toward guaiacol oxidation are quite different; the iron corrole reconstituted HRP decelerates H2O2-dependent oxidation of guaiacol, while the same reaction catalyzed by iron corrole reconstituted Mb has the opposite effect and accelerates the reaction. This finding can be attributed to the difference in the oxidation states of the corrole iron when these proteins are in the resting state.

Hayashi, T; Oohora, K.

Inorg. Chem. 2020, 59, 11995-12004, 10.1021/acs.inorgchem.0c00901

Methyl-coenzyme M reductase (MCR), which contains the nickel hydrocorphinoid cofactor F430, is responsible for biological methane generation under anaerobic conditions via a reaction mechanism which has not been completely elucidated. In this work, myoglobin reconstituted with an artificial cofactor, nickel(I) tetradehydrocorrin (NiI(TDHC)), is used as a protein-based functional model for MCR. The reconstituted protein, rMb(NiI(TDHC)), is found to react with methyl donors such as methyl p-toluenesulfonate and trimethylsulfonium iodide with methane evolution observed in aqueous media containing dithionite. Moreover, rMb(NiI(TDHC)) is found to convert benzyl bromide derivatives to reductively debrominated products without homocoupling products. The reactivity increases in the order of primary > secondary > tertiary benzylic carbons, indicating steric effects on the reaction of the nickel center with the benzylic carbon in the initial step. In addition, Hammett plots using a series of para-substituted benzyl bromides exhibit enhancement of the reactivity with introduction of electron-withdrawing substituents, as shown by the positive slope against polar substituent constants. These results suggest a nucleophilic SN2-type reaction of the Ni(I) species with the benzylic carbon to provide an organonickel species as an intermediate. The reaction in D2O buffer at pD 7.0 causes a complete isotope shift of the product by +1 mass unit, supporting our proposal that protonation of the organonickel intermediate occurs during product formation. Although the turnover numbers are limited due to inactivation of the cofactor by side reactions, the present findings will contribute to elucidating the reaction mechanism of MCR-catalyzed methane generation from activated methyl sources and dehalogenation.

Hilvert, D.

Angew. Chem. Int. Ed. 2021, 60, 15063-15068, 10.1002/anie.202103437

Changing the primary metal coordination sphere is a powerful strategy for tuning metalloprotein properties. Here we used amber stop codon suppression with engineered pyrrolysyl-tRNA synthetases, including two newly evolved enzymes, to replace the proximal histidine in myoglobin with Nδ-methylhistidine, 5-thiazoylalanine, 4-thiazoylalanine and 3-(3-thienyl)alanine. In addition to tuning the heme redox potential over a >200 mV range, these noncanonical ligands modulate the protein's carbene transfer activity with ethyl diazoacetate. Variants with increased reduction potential proved superior for cyclopropanation and N–H insertion, whereas variants with reduced Eo values gave higher S–H insertion activity. Given the functional importance of histidine in many enzymes, these genetically encoded analogues could be valuable tools for probing mechanism and enabling new chemistries.

Lu, Y.

Chem. - Eur. J. 2009, 15, 7481-7489, 10.1002/chem.200802449

To demonstrate protein modulation of metal‐cofactor reactivity through noncovalent interactions, pH‐dependent sulfoxidation and 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulphonic acid) (ABTS) oxidation reactivity of a designed myoglobin (Mb) containing non‐native Mn–salen complex (1) was investigated using H2O2 as the oxidant. Incorporation of 1 inside the Mb resulted in an increase in the turnover numbers through exclusion of water from the metal complex and prevention of Mn–salen dimer formation. Interestingly, the presence of protein in itself is not enough to confer the increase activity as mutation of the distal His64 in Mb to Phe to remove hydrogen‐bonding interactions resulted in no increase in the turnover numbers, while mutation His64 to Arg, another residue with ability to hydrogen‐bond interactions, resulted in an increase in reactivity. These results strongly suggest that the distal ligand His64, through its hydrogen‐bonding interaction, plays important roles in enhancing and fine‐tuning reactivity of the Mn–salen complex. Nonlinear least‐squares fitting of rate versus pH plots demonstrates that 1⋅Mb(H64X) (X=H, R and F) and the control Mn–salen 1 exhibit pKa values varying from pH 6.4 to 8.3, and that the lower pKa of the distal ligand in 1⋅Mb(H64X), the higher the reactivity it achieves. Moreover, in addition to the pKa at high pH, 1⋅Mb displays another pKa at low pH, with pKa of 5.0±0.08. A comparison of the effect of different pH on sulfoxidation and ABTS oxidation indicates that, while the intermediate produced at low pH conditions could only perform sulfoxidation, the intermediate at high pH could oxidize both sulfoxides and ABTS. Such a fine‐control of reactivity through hydrogen‐bonding interactions by the distal ligand to bind, orient and activate H2O2 is very important for designing artificial enzymes with dramatic different and tunable reactivity from catalysts without protein scaffolds.

J. Am. Chem. Soc. 1999, 121, 7747-7750, 10.1021/ja9841005

Peroxidase activity of a myoglobin reconstituted with a chemically modified heme 1 is reported. The heme 1 bearing a total of eight carboxylates bound to the terminal of propionate side chains is incorporated into apomyoglobin from horse heart to obtain a new reconstituted myoglobin, rMb(1), with a unique binding domain structure. The UV−vis, CD, and NMR spectra of rMb(1) are comparable with those of native myoglobin, nMb. The mixing of rMb(1) with hydrogen peroxide yields a peroxidase compound II-like species, rMb(1)-II, since the spectrum of rMb(1)-II is identical with that observed for nMb. Stoichiometric oxidation of several small molecules by rMb(1)-II, demonstrates the significant reactivity. (i) The oxidation of cationic substrate such as [Ru(NH3)6]2+ by rMb(1)-II is faster than that observed for oxoferryl species of nMb, nMb-II. (ii) Anionic substrates such as ferrocyanide are unsuitable for the oxidation by rMb(1)-II. (iii) Oxidations of catechol, hydroquinone, and guaiacol are dramatically enhanced by rMb(1)-II (14−32-fold) compared to those observed for nMb-II. Thus, the chemical modification of heme-propionates can alter substrate specificity. Steady-state kinetic measurements indicate that both the reactivity and substrate affinity toward guaiacol oxidation by rMb(1) are improved, so that the specificity, kcat/Km, is 13-fold higher than that in nMb. This result strongly suggests that the artificially modified heme-propionates may increase the accessibility of neutral aromatic substrates to the heme active site. The present work demonstrates that the chemical mutation of prosthetic group is a new strategy to create proteins with engineered function.

Matsuo, T.

Chem. - Asian J. 2011, 6, 2491-2499, 10.1002/asia.201100107

H64D myoglobin mutant was reconstituted with two different types of synthetic hemes that have aromatic rings and a carboxylate‐based cluster attached to the terminus of one or both of the heme‐propionate moieties, thereby forming a “single‐winged cofactor” and “double‐winged cofactor,” respectively. The reconstituted mutant myoglobins have smaller Km values with respect to 2‐methoxyphenol oxidation activity relative to the parent mutant with native heme. This suggests that the attached moiety functions as a substrate‐binding domain. However, the kcat value of the mutant myoglobin with the double‐winged cofactor is much lower than that of the mutant with the native heme. In contrast, the mutant reconstituted with the single‐winged cofactor has a larger kcat value, thereby resulting in overall catalytic activity that is essentially equivalent to that of the native horseradish peroxidase. Enhanced peroxygenase activity was also observed for the mutant myoglobin with the single‐winged cofactor, thus indicating that introduction of an artificial substrate‐binding domain at only one of the heme propionates in the H64D mutant is the optimal engineering strategy for improving the peroxidase activity of myoglobin.

Watanabe, Y.

Angew. Chem. Int. Ed. 2003, 42, 1005-1008, 10.1002/anie.200390256

Insertion of a symmetric metal complex, [CrIII(5,5′‐tBu‐salophen)]+ (H2salophen=N,N′‐bis(salicylidene)‐1,2‐phenylenediamine), into the active site of apomyoglobin is demonstrated (see picture). The metal ion and the ligand structure are very important factors that influence the binding affinity of the metal complex with the myoglobin (Mb) cavity. Semisynthetic metalloenzymes can catalyze enantioselective sulfoxidation by using the chiral protein cavity.

Lu, Y.

Chem. Commun. 2008, 1665, 10.1039/b718915j

We demonstrate that incorporation of MnSalen into a protein scaffold enhances the chemoselectivity in sulfoxidation of thioanisole and find that both the polarity and hydrogen bonding of the protein scaffold play an important role in tuning the chemoselectivity.

Ghirlanda, G.

Chem. Commun. 2014, 50, 15852-15855, 10.1039/c4cc06700b

Hydrogen is an attractive fuel with potential for production scalability, provided that inexpensive, efficient molecular catalysts utilizing base metals can be developed for hydrogen production. Here we show for the first time that cobalt myoglobin (CoMyo) catalyzes hydrogen production in mild aerobic conditions with turnover number of 520 over 8 hours. Compared to free Co-protoporphyrin IX, incorporation into the myoglobin scaffold results in a 4-fold increase in photoinduced hydrogen production activity. Engineered variants in which specific histidine resides in proximity of the active site were mutated to alanine result in modulation of the catalytic activity, with the H64A/H97A mutant displaying activity 2.5-fold higher than wild type. Our results demonstrate that protein scaffolds can augment and modulate the intrinsic catalytic activity of molecular hydrogen production catalysts.

Lin, Y.-W.

ACS Catal. 2020, 10, 14359-14365, 10.1021/acscatal.0c04572

Design of artificial nucleases is essential in biotechnology and biomedicine, whereas few artificial nucleases can both cleave and degrade DNA molecules. Heme proteins are potential enzymes for DNA cleavage. Using a small heme protein, myoglobin (Mb), as a model protein, we engineered a metal-binding motif of [1-His-1-Glu] (native His64 and mutated Glu29) in the heme distal site. The single mutant of L29E Mb was capable of not only efficient DNA cleavage but also DNA degradation upon Mg2+ binding to the heme distal site, as shown by an X-ray crystal structure of the Mg2+-L29E Mb complex. Molecular docking of the protein–DNA complex revealed multiple hydrogen-bonding interactions at their interfaces, involving both minor and major grooves of DNA. Moreover, both the distal Arg45 and the ligand Glu29 were identified as critical residues for the nuclease activity. This study reports the structure of a water-bridged heterodinuclear center of Mg-heme (Mg2+-H2O-Fe3+), showing a similar function as the homodinuclear center (MgA2+-H2O–MgB2+) in natural nuclease, which indicates that the Mg2+-L29E Mb complex is an effective artificial nuclease.