33 publications

33 publications

Abiological Catalysis by Artificial Haem Proteins Containing Noble Metals in Place of Iron

Hartwig, J. F.

Nature, 2016, 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.


Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Myoglobin (Mb)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 7260
ee: 68
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Myoglobin (Mb)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 92
ee: 84
PDB: ---
Notes: ---

Addressable DNA–Myoglobin Photocatalysis

Niemeyer, C. M.

Chem. - Asian J., 2009, 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.


Metal: Ru
Ligand type: Bipyridine
Host protein: Myoglobin (Mb)
Anchoring strategy: Supramolecular
Optimization: ---
Reaction: Photooxidation
Max TON: ---
ee: ---
PDB: ---
Notes: Horse heart myoglobin

A Designed Functional Metalloenzyme that Reduces O2 to H2O with Over One Thousand Turnovers

Lu, Y.

Angew. Chem., Int. Ed., 2012, 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.


Metal: Cu
Ligand type: Amino acid
Host protein: Myoglobin (Mb)
Anchoring strategy: Dative
Optimization: Chemical & genetic
Max TON: 1056
ee: ---
PDB: 4FWX
Notes: Sperm whale myoglobin

A Designed Metalloenzyme Achieving the Catalytic Rate of a Native Enzyme

Lu, Y.; Wang, J.

J. Am. Chem. Soc., 2015, 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.


Metal: Cu
Ligand type: Amino acid
Host protein: Myoglobin (Mb)
Anchoring strategy: Dative
Optimization: Genetic
Reaction: O2 reduction
Max TON: ---
ee: ---
PDB: ---
Notes: O2 reduction rates of 52 s-1 were achieved in combination with the native redox partner cyt b5.

A Noncanonical Proximal Heme Ligand Affords an Efficient Peroxidase in a Globin Fold

Green, A. P.; Hilvert, D.

J. Am. Chem. Soc., 2018, 10.1021/jacs.7b12621


Metal: Fe
Host protein: Myoglobin (Mb)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Oxidation
Max TON: ~1650
ee: ---
PDB: 5OJ9
Notes: Oxidation of amplex red

A Site-Selective Dual Anchoring Strategy for Artificial Metalloprotein Design

Lu, Y.

J. Am. Chem. Soc., 2004, 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.


Metal: Mn
Ligand type: Salen
Host protein: Myoglobin (Mb)
Anchoring strategy: Covalent
Optimization: Genetic
Reaction: Sulfoxidation
Max TON: 3.9
ee: 51
PDB: 1MBO
Notes: Sperm whale myoglobin

Bimetallic Copper-Heme-Protein-DNA Hybrid Catalyst for Diels Alder Reaction

Fruk, L.; Niemeyer, C. M.

Croat. Chem. Acta, 2011, 10.5562/cca1828


Metal: Cu
Ligand type: Bipyridine
Host protein: Myoglobin (Mb)
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: 7.1
ee: 18
PDB: ---
Notes: Horse heart myoglobin

Capture and Characterization of a Reactive Haem– Carbenoid Complex in an Artificial Metalloenzyme

Hilvert, D.

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


Metal: Fe
Host protein: Myoglobin (Mb)
Anchoring strategy: ---
Optimization: Genetic
Reaction: Cyclopropanation
Max TON: 1000
ee: 99
PDB: 6F17
Notes: Structure of the Mb*(NMH) haem-iron complex

Metal: Fe
Host protein: Myoglobin (Mb)
Anchoring strategy: ---
Optimization: Genetic
Reaction: Cyclopropanation
Max TON: 1000
ee: 99
PDB: 6G5B
Notes: Structure of the Mb*(NMH) haem-iron–carbenoid complex

Catalytic Cyclopropanation by Myoglobin Reconstituted with Iron Porphycene: Acceleration of Catalysis due to Rapid Formation of the Carbene Species

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

J. Am. Chem. Soc., 2017, 10.1021/jacs.7b10154


Metal: Fe
Ligand type: Amino acid; Porphycene
Host protein: Myoglobin (Mb)
Anchoring strategy: Reconstitution
Optimization: ---
Reaction: Cyclopropanation
Max TON: ---
ee: ---
PDB: ---
Notes: Cyclopropanation of styrene with ethyl diazoacetate: kcat/KM = 1.3 mM-1 * s-1, trans/cis = 99:1

Catalytic Reduction of NO to N2O by a Designed Heme Copper Center in Myoglobin: Implications for the Role of Metal Ions

Lu, Y.

J. Am. Chem. Soc., 2006, 10.1021/ja058822p


Metal: Cu
Ligand type: Amino acid; Porphyrin
Host protein: Myoglobin (Mb)
Anchoring strategy: Dative
Optimization: Genetic
Max TON: 2400
ee: ---
PDB: ---
Notes: Sperm whale myoglobin

Cobaloxime-Based Artificial Hydrogenase

Artero, V.

Inorg. Chem., 2014, 10.1021/ic501014c


Metal: Co
Ligand type: Oxime
Host protein: Myoglobin (Mb)
Anchoring strategy: Supramolecular
Optimization: Chemical
Reaction: H2 evolution
Max TON: 5
ee: ---
PDB: ---
Notes: Sperm whale myoglobin

Coordinated Design of Cofactor and Active Site Structures in Development of New Protein Catalysts

Watanabe, Y.

J. Am. Chem. Soc., 2005, 10.1021/ja045995q


Metal: Mn
Ligand type: Salophen
Host protein: Myoglobin (Mb)
Anchoring strategy: Reconstitution
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: 1V9Q
Notes: ---

Metal: Cr
Ligand type: Salophen
Host protein: Myoglobin (Mb)
Anchoring strategy: Reconstitution
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: 1J3F
Notes: ---

Metal: Mn
Ligand type: Salen
Host protein: Myoglobin (Mb)
Anchoring strategy: Reconstitution
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Metal: Cr
Ligand type: Salen
Host protein: Myoglobin (Mb)
Anchoring strategy: Reconstitution
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Crystal Structure and Peroxidase Activity of Myoglobin Reconstituted with Iron Porphycene

Inorg. Chem., 2006, 10.1021/ic061130x


Metal: Fe
Ligand type: Porphycene
Host protein: Myoglobin (Mb)
Anchoring strategy: Reconstitution
Optimization: ---
Max TON: ---
ee: ---
PDB: 1MBI
Notes: ---

C(sp3)–H Bond Hydroxylation Catalyzed by Myoglobin Reconstituted with Manganese Porphycene

Hayashi, T

J. Am. Chem. Soc., 2013, 10.1021/ja409404k


Metal: Mn
Ligand type: Porphycene
Host protein: Myoglobin (Mb)
Anchoring strategy: Reconstitution
Optimization: ---
Reaction: Hydroxylation
Max TON: ---
ee: ---
PDB: 2WI8
Notes: ---

Defining the Role of Tyrosine and Rational Tuning of Oxidase Activity by Genetic Incorporation of Unnatural Tyrosine Analogs

Lu, Y.; Wang, J.

J. Am. Chem. Soc., 2015, 10.1021/ja5109936


Metal: Cu
Ligand type: Porphyrin
Host protein: Myoglobin (Mb)
Anchoring strategy: Dative
Optimization: Chemical & genetic
Max TON: 1200
ee: ---
PDB: 4FWX
Notes: Sperm whale myoglobin

Enzyme stabilization via computationally guided protein stapling

Fasan, R.; Khare, S. D.

Proc. Natl. Acad. Sci. U. S. A., 2017, 10.1073/pnas.1708907114


Metal: Fe
Ligand type: Porphyrin
Host protein: Myoglobin (Mb)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Cyclopropanation
Max TON: 4740
ee: 99.2
PDB: ---
Notes: Stapling of protein via thioether bond formation between the noncanonical amino acid O-2-bromoethyl tyrosine and cysteine

Hybridization of Modified-Heme Reconstitution and Distal Histidine Mutation to Functionalize Sperm Whale Myoglobin

J. Am. Chem. Soc., 2004, 10.1021/ja038798k


Metal: Fe
Host protein: Myoglobin (Mb)
Anchoring strategy: Reconstitution
Optimization: Genetic
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Intramolecular C(sp3)-H Amination of Arylsulfonyl Azides with Engineered and Artificial Myoglobin-Based Catalysts

Fasan, R.

Bioorg. Med. Chem., 2014, 10.1016/j.bmc.2014.05.015


Metal: Mn
Ligand type: Amino acid; Porphyrin
Host protein: Myoglobin (Mb)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 142
ee: ---
PDB: ---
Notes: ---

Introducing a 2-His-1-Glu Nonheme Iron Center into Myoglobin Confers Nitric Oxide Reductase Activity

Lu, Y.

J. Am. Chem. Soc., 2010, 10.1021/ja103516n


Metal: Fe
Ligand type: Amino acid
Host protein: Myoglobin (Mb)
Anchoring strategy: Dative
Optimization: Genetic
Max TON: 320
ee: ---
PDB: 3MN0
Notes: Sperm whale myoglobin

Manganese(V) Porphycene Complex Responsible for Inert C–H Bond Hydroxylation in a Myoglobin Matrix

Oohora, K.

J. Am. Chem. Soc., 2017, 10.1021/jacs.7b11288


Metal: Mn
Ligand type: Amino acid; Porphycene
Host protein: Myoglobin (Mb)
Anchoring strategy: Reconstitution
Optimization: ---
Reaction: Hydroxylation
Max TON: 13
ee: ---
PDB: 5YL3
Notes: ---

Meso-Unsubstituted Iron Corrole in Hemoproteins: Remarkable Differences in Effects on Peroxidase Activities between Myoglobin and Horseradish Peroxidase

J. Am. Chem. Soc., 2009, 10.1021/ja907428e


Metal: Fe
Ligand type: Corrole
Host protein: Myoglobin (Mb)
Anchoring strategy: Reconstitution
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Metal: Fe
Ligand type: Corrole
Anchoring strategy: Reconstitution
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Noncovalent Modulation of pH-Dependent Reactivity of a Mn–Salen Cofactor in Myoglobin with Hydrogen Peroxide

Lu, Y.

Chem. - Eur. J., 2009, 10.1002/chem.200802449


Metal: Mn
Ligand type: Salen
Host protein: Myoglobin (Mb)
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Reaction: Sulfoxidation
Max TON: 4.1
ee: 50
PDB: ---
Notes: Sperm whale myoglobin

Peroxidase Activity of Myoglobin is Enhanced by Chemical Mutation of Heme-Propionates

J. Am. Chem. Soc., 1999, 10.1021/ja9841005


Metal: Fe
Host protein: Myoglobin (Mb)
Anchoring strategy: Reconstitution
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Precise Design of Artificial Cofactors for Enhancing Peroxidase Activity of Myoglobin: Myoglobin Mutant H64D Reconstituted with a “Single-Winged Cofactor” is Equivalent to Native Horseradish Peroxidase in Oxidation Activity

Matsuo, T.

Chem. - Asian J., 2011, 10.1002/asia.201100107


Metal: Fe
Host protein: Myoglobin (Mb)
Anchoring strategy: Reconstitution
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Preparation of Artificial Metalloenzymes by Insertion of Chromium(III) Schiff Base Complexes into apo-Myoglobin Mutants

Watanabe, Y.

Angew. Chem., Int. Ed., 2003, 10.1002/anie.200390256


Metal: Cr
Ligand type: Salophen
Host protein: Myoglobin (Mb)
Anchoring strategy: Reconstitution
Optimization: Genetic
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Protein Scaffold of a Designed Metalloenzyme Enhances the Chemoselectivity in Sulfoxidation of Thioanisole

Lu, Y.

Chem. Commun., 2008, 10.1039/b718915j


Metal: Mn
Ligand type: Salen
Host protein: Myoglobin (Mb)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Sulfoxidation
Max TON: 5.2
ee: 60
PDB: ---
Notes: Sperm whale myoglobin

Protein Secondary-Shell Interactions Enhance the Photoinduced Hydrogen Production of Cobalt Protoporphyrin IX

Ghirlanda, G.

Chem. Commun., 2014, 10.1039/c4cc06700b


Metal: Co
Ligand type: Porphyrin
Host protein: Myoglobin (Mb)
Anchoring strategy: Metal substitution
Optimization: Genetic
Reaction: H2 evolution
Max TON: 518
ee: ---
PDB: ---
Notes: ---

Rational Design of a Structural and Functional Nitric Oxide Reductase

Lu, Y.

Nature, 2009, 10.1038/nature08620


Metal: Fe
Ligand type: Amino acid
Host protein: Myoglobin (Mb)
Anchoring strategy: Dative
Optimization: Genetic
Reaction: NO reduction
Max TON: ~5
ee: ---
PDB: 3K9Z
Notes: Design of a catalytically active non-haem iron-binding site (FeB) in sperm whale myoglobin.

Roles of Glutamates and Metal Ions in a Rationally Designed Nitric Oxide Reductase Based on Myoglobin

Lu, Y.

Proc. Natl. Acad. Sci. U. S. A., 2010, 10.1073/pnas.1000526107


Metal: Fe
Ligand type: Amino acid
Host protein: Myoglobin (Mb)
Anchoring strategy: Dative
Optimization: Genetic
Reaction: NO reduction
Max TON: ---
ee: ---
PDB: 3M39
Notes: X-ray structure of mutant I107E.

Metal: Cu
Ligand type: Amino acid
Host protein: Myoglobin (Mb)
Anchoring strategy: Dative
Optimization: Genetic
Reaction: NO reduction
Max TON: ---
ee: ---
PDB: 3M3A
Notes: X-ray structure of mutant I107E.

Significant Improvement of Oxidase Activity Through the Genetic Incorporation of a Redox-Active Unnatural Amino Acid

Lu, Y.; Wang, J.

Chem. Sci., 2015, 10.1039/C5SC01126D


Metal: Cu
Ligand type: Amino acid
Host protein: Myoglobin (Mb)
Anchoring strategy: Dative
Optimization: Genetic
Reaction: O2 reduction
Max TON: >1100
ee: ---
PDB: ---
Notes: Reduction potential was lowered by incorporation of the unnatural amino acid 3-methoxy tyrosine.