24 publications

24 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: ---

An Artificial Metalloenzyme with the Kinetics of Native Enzymes

Hartwig, J. F.

Science, 2016, 10.1126/science.aah4427

Natural enzymes contain highly evolved active sites that lead to fast rates and high selectivities. Although artificial metalloenzymes have been developed that catalyze abiological transformations with high stereoselectivity, the activities of these artificial enzymes are much lower than those of natural enzymes. Here, we report a reconstituted artificial metalloenzyme containing an iridium porphyrin that exhibits kinetic parameters similar to those of natural enzymes. In particular, variants of the P450 enzyme CYP119 containing iridium in place of iron catalyze insertions of carbenes into C–H bonds with up to 98% enantiomeric excess, 35,000 turnovers, and 2550 hours−1 turnover frequency. This activity leads to intramolecular carbene insertions into unactivated C–H bonds and intermolecular carbene insertions into C–H bonds. These results lift the restrictions on merging chemical catalysis and biocatalysis to create highly active, productive, and selective metalloenzymes for abiological reactions.


Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Cytochrome P450 (CYP119)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 582
ee: 98
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Cytochrome P450 (CYP119)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 35129
ee: 91
PDB: ---
Notes: ---

A Well-Defined Osmium–Cupin Complex: Hyperstable Artificial Osmium Peroxygenase

Fujieda, N.; Itoh, S.

J. Am. Chem. Soc., 2017, 10.1021/jacs.7b00675


Metal: Os
Ligand type: Amino acid
Host protein: TM1459 cupin
Anchoring strategy: Metal substitution
Optimization: Genetic
Reaction: Dihydroxylation
Max TON: 45
ee: ---
PDB: 5WSE
Notes: Exclusively cis dihydroxylation product obtained

Metal: Os
Ligand type: Amino acid
Host protein: TM1459 cupin
Anchoring strategy: Metal substitution
Optimization: Genetic
Reaction: Dihydroxylation
Max TON: 45
ee: ---
PDB: 5WSF
Notes: Exclusively cis dihydroxylation product obtained

Beyond Iron: Iridium-Containing P450 Enzymes for Selective Cyclopropanations of Structurally Diverse Alkenes

Hartwig, J. F.

ACS Cent. Sci., 2017, 10.1021/acscentsci.6b00391


Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Cytochrome P450 (CYP119)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: Cyclopropanation
Max TON: 10181
ee: 98
PDB: ---
Notes: Selectivity for cis product (cis/trans = 90:1)

Catalytic Properties and Specificity of the Extracellular Nuclease of Staphylococcus Aureus

Cuatrecasas, P.

J. Biol. Chem., 1967, PMID 4290246


Metal: Sr
Ligand type: Amino acid
Host protein: Nuclease from S. aureus
Anchoring strategy: Metal substitution
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: DNA cleavage

Catalytic Water Oxidation by Iridium-Modified Carbonic Anhydrase

Lee, S.-Y.

Chem. - Asian J., 2017, 10.1002/asia.201701543


Metal: Ir
Ligand type: Amino acid
Anchoring strategy: Metal substitution
Optimization: Chemical
Reaction: Water oxidation
Max TON: ---
ee: ---
PDB: ---
Notes: Sodium periodate as sacrificial oxidant. TOF at pH 7 and 30°C is 39.8 min-1.

Chemoselective, Enzymatic C−H Bond Amination Catalyzed by a Cytochrome P450 Containing an Ir(Me)-PIX Cofactor

Hartwig, J. F.

J. Am. Chem. Soc., 2017, 10.1021/jacs.6b11410


Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Cytochrome P450 (CYP119)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 294
ee: 26
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Cytochrome P450 (CYP119)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 192
ee: 95
PDB: ---
Notes: ---

Direct Hydrogenation of Carbon Dioxide by an Artificial Reductase Obtained by Substituting Rhodium for Zinc in the Carbonic Anhydrase Catalytic Center. A Mechanistic Study

Marino, T.

ACS Catal., 2015, 10.1021/acscatal.5b00185


Metal: Rh
Ligand type: Amino acid
Anchoring strategy: Metal substitution
Optimization: ---
Reaction: Hydrogenation
Max TON: ---
ee: ---
PDB: ---
Notes: Computational study of the reaction mechanism of the formation of HCOOH from CO2

Going Beyond Structure: Nickel-Substituted Rubredoxin as a Mechanistic Model for the [NiFe] Hydrogenases

Shafaat, H. S.

J. Am. Chem. Soc., 2018, 10.1021/jacs.8b05194


Metal: Ni
Ligand type: Amino acid
Host protein: Rubredoxin (Rd)
Anchoring strategy: Metal substitution
Optimization: Genetic
Reaction: H2 evolution
Max TON: ---
ee: ---
PDB: ---
Notes: TOF = 149 s-1

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: ---

Manganese-Substituted Carbonic Anhydrase as a New Peroxidase

Kazlauskas, R. J.

Chem. - Eur. J., 2006, 10.1002/chem.200501413


Metal: Mn
Ligand type: Amino acid
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: Epoxidation
Max TON: 22
ee: 67
PDB: ---
Notes: ---

Metal: Mn
Ligand type: Amino acid
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: Epoxidation
Max TON: 9.5
ee: 55
PDB: 4CAC
Notes: PDB ID 4CAC = Structure of Zn containing hCAII

Metal Ion Dependent Binding of Sulphonamide to Carbonic Anhydrase

Coleman, J. E.

Nature, 1967, 10.1038/214193a0


Metal: Co
Ligand type: Amino acid
Host protein: Human carbonic anhydrase
Anchoring strategy: Metal substitution
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: CO2 hydration

Metal: Co
Ligand type: Amino acid
Host protein: Human carbonic anhydrase
Anchoring strategy: Metal substitution
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: Ester cleavage

Metal Substitution in Thermolysin: Catalytic Properties of Tungstate Thermolysin in Sulfoxidation with H2O2

Sheldon, R. A.

Can. J. Chem., 2002, 10.1139/v02-082


Metal: W
Ligand type: Amino acid
Host protein: Thermolysin
Anchoring strategy: Metal substitution
Optimization: Chemical
Reaction: Sulfoxidation
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Multifunctional Nanoenzymes from Carbonic Anhydrase Skeleton

Yilmaz, F.

Process Biochem., 2018, 10.1016/j.procbio.2018.06.005


Metal: Zn
Ligand type: Amino acid
Host protein: Carbonic anhydrase (CA)
Anchoring strategy: Metal substitution
Optimization: Chemical
Reaction: Hydrolysis
Max TON: ---
ee: ---
PDB: ---
Notes: Cross-linked carbonic anhydrase nano-enzyme particles (93 nm in diameter). Hydrolysis of 4-nitrophenyl acetate.

Metal: Rh
Ligand type: Amino acid
Host protein: Carbonic anhydrase (CA)
Anchoring strategy: Metal substitution
Optimization: Chemical
Reaction: Hydration
Max TON: ---
ee: ---
PDB: ---
Notes: Cross-linked carbonic anhydrase nano-enzyme particles (93 nm in diameter). Hydration of styrene.

Metal: Mn
Ligand type: Amino acid
Host protein: Carbonic anhydrase (CA)
Anchoring strategy: Metal substitution
Optimization: Chemical
Reaction: Oxidation
Max TON: ---
ee: ---
PDB: ---
Notes: Cross-linked carbonic anhydrase nano-enzyme particles (93 nm in diameter). Oxidation of styrene.

Nickel-Substituted Rubredoxin as a Minimal Enzyme Model for Hydrogenase

Shafaat, H. S.

J. Phys. Chem. Lett., 2015, 10.1021/acs.jpclett.5b01750


Metal: Ni
Ligand type: Tetrathiolate
Host protein: Rubredoxin (Rd)
Anchoring strategy: Metal substitution
Optimization: ---
Reaction: H2 evolution
Max TON: 300
ee: ---
PDB: ---
Notes: ---

Polymer Enzyme Conjugates as Chiral Ligands for Sharpless Dihydroxylation of Alkenes in Organic Solvents

Tiller, J. C.

ChemBioChem, 2015, 10.1002/cbic.201402339


Metal: Os
Ligand type: Amino acid
Host protein: Laccase
Anchoring strategy: Metal substitution
Optimization: Chemical
Reaction: Dihydroxylation
Max TON: 80
ee: 98
PDB: ---
Notes: ---

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: ---

Rare Earth Metal Ions as Probes of Calcium Binding Sites in Proteins: Neodynium Acceleration of the Activation of Trypsinogen

Birnbaum, E. R.; Darnall, D. W.

J. Biol. Chem., 1970, PMID 5484822


Metal: Nd
Ligand type: Amino acid
Host protein: Trypsin
Anchoring strategy: Metal substitution
Optimization: ---
Max TON: <1
ee: ---
PDB: ---
Notes: ---

Reengineering Cyt b562 for Hydrogen Production: A Facile Route to Artificial Hydrogenases

Ghirlanda, G.

Biochim. Biophys. Acta, Bioenerg., 2016, 10.1016/j.bbabio.2015.09.001


Metal: Co
Ligand type: Porphyrin
Host protein: Cytochrome b562
Anchoring strategy: Metal substitution
Optimization: Genetic
Reaction: H2 evolution
Max TON: 1450
ee: ---
PDB: ---
Notes: ---

Regioselective Hydroformylation of Styrene Using Rhodium-Substituted Carbonic Anhydrase

Kazlauskas, R. J.

ChemCatChem, 2010, 10.1002/cctc.201000159


Metal: Rh
Ligand type: Acac; Carbonyl
Anchoring strategy: Metal substitution
Optimization: Genetic
Reaction: Hydroformylation
Max TON: 298
ee: ---
PDB: 4CAC
Notes: PDB ID 4CAC = Structure of Zn containing hCAII

Semisynthetic and Biomolecular Hydrogen Evolution Catalysts

Bren, K. L.

Inorg. Chem., 2016, 10.1021/acs.inorgchem.5b02054


Metal: Co
Ligand type: Porphyrin
Host protein: Cytochrome c552
Anchoring strategy: Metal substitution
Optimization: Genetic
Reaction: H2 evolution
Max TON: 27000
ee: ---
PDB: ---
Notes: Electrocatalysis

Stereoselective Hydrogenation of Olefins Using Rhodium-Substituted Carbonic Anhydrase—A New Reductase

Kazlauskas, R. J.

Chem. - Eur. J., 2008, 10.1002/chem.200801673


Metal: Rh
Ligand type: COD
Anchoring strategy: Metal substitution
Optimization: Genetic
Reaction: Hydrogenation
Max TON: 15.8
ee: ---
PDB: ---
Notes: ---

Metal: Rh
Ligand type: COD
Anchoring strategy: Metal substitution
Optimization: Genetic
Reaction: Hydrogenation
Max TON: 80.5
ee: ---
PDB: 4CAC
Notes: PDB ID 4CAC = Structure of Zn containing hCAII

Studies on the Oxidase Activity of Copper (II) Carboxypeptidase A

Kaiser, E. T.

J. Chem. Soc., Chem. Commun., 1976, 10.1039/C39760000830


Metal: Cu
Ligand type: Amino acid
Host protein: Carboxypeptidase A
Anchoring strategy: Metal substitution
Optimization: ---
Reaction: Oxidation
Max TON: ---
ee: ---
PDB: ---
Notes: Oxidation of vitamin C

Transforming Carbonic Anhydrase into Epoxide Synthase by Metal Exchange

Soumillion, P.

ChemBioChem, 2006, 10.1002/cbic.200600127


Metal: Mn
Ligand type: Amino acid
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: Epoxidation
Max TON: 4.1
ee: 52
PDB: ---
Notes: ---

Metal: Mn
Ligand type: Amino acid
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: Epoxidation
Max TON: 10.3
ee: 40
PDB: ---
Notes: ---