Rimoldi, I.

New J. Chem. 2018, 42, 18773-18776, 10.1039/C8NJ04558E

The imine reductase formed by the (R)-CAMPY ligand bound to the S112M Sav mutant showed an 83% ee in the asymmetric transfer hydrogenation of 6,7-dimethoxy-1-methyl-3,4-dihydroisoquinoline.

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.

Mahy, J.-P.

Eur. J. Biochem. 1998, 257, 121-130, 10.1046/j.1432-1327.1998.2570121.x

The topology of the binding site has been studied for two monoclonal antibodies 13G10 and 14H7, elicited against iron(III)‐α,α,α,β‐meso‐tetrakis(ortho‐carboxyphenyl)porphyrin {α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin]}, and which exhibit in the presence of this α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] cofactor a peroxidase activity. A comparison of the dissociation constants of the complexes of 13G10 and 14H7 with various tetra‐aryl‐substituted porphyrin has shown that : (a) the central iron(III) atom of α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] is not recognized by either of the two antibodies; and (b) the ortho‐carboxylate substituents of the meso‐phenyl rings of α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] are essential for the recognition of the porphyrin by 13G10 and 14H7. Measurement of the dissociation constants for the complexes of 13G10 and 14H7 with the four atropoisomers of (o‐COOHPh)4‐porphyrinH2 as well as mono‐ and di‐ortho‐carboxyphenyl‐substituted porphyrins suggests that the three carboxylates in the α, α, β position are recognized by both 13G10 and 14H7 with the two in the α, β positions more strongly bound to the antibody protein. Accordingly, the topology of the active site of 13G10 and 14H7 has roughly two‐thirds of the α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] cofactor inserted into the binding site of the antibodies, with one of the aryl ring remaining outside. Three of the carboxylates are bound to the protein but no amino acid residue acts as an axial ligand to the iron atom. Chemical modification of lysine, histidine, tryptophan and arginine residues has shown that only modification of arginine residues causes a decrease in both the binding of α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] and the peroxidase activity of both antibodies. Consequently, at least one of the carboxylates of the hapten is bound to an arginine residue and no amino acids such as lysine, histidine or tryptophan participate in the catalysis of the heterolytic cleavage of the O‐O bond of H2O2. In addition, the amino acid sequence of both antibodies not only reveals the presence of arginine residues, which could be those involved in the binding of the carboxylates of the hapten, but also the presence of several amino acids in the complementary determining regions which could bind other carboxylates through a network of H bonds.

Pecoraro, V.L.

Angew. Chem. Int. Ed. 2014, 53, 7900-7903, 10.1002/anie.201404925

Protein design will ultimately allow for the creation of artificial enzymes with novel functions and unprecedented stability. To test our current mastery of nature’s approach to catalysis, a ZnII metalloenzyme was prepared using de novo design. α3DH3 folds into a stable single‐stranded three‐helix bundle and binds ZnII with high affinity using His3O coordination. The resulting metalloenzyme catalyzes the hydration of CO2 better than any small molecule model of carbonic anhydrase and with an efficiency within 1400‐fold of the fastest carbonic anhydrase isoform, CAII, and 11‐fold of CAIII.

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.

Science 2018, 361, 1098-1101, 10.1126/science.aat8474

Multielectron redox reactions often require multicofactor metalloenzymes to facilitate coupled electron and proton movement, but it is challenging to design artificial enzymes to catalyze these important reactions, owing to their structural and functional complexity. We report a designed heteronuclear heme-[4Fe-4S] cofactor in cytochrome c peroxidase as a structural and functional model of the enzyme sulfite reductase. The initial model exhibits spectroscopic and ligand-binding properties of the native enzyme, and sulfite reduction activity was improved—through rational tuning of the secondary sphere interactions around the [4Fe-4S] and the substrate-binding sites—to be close to that of the native enzyme. By offering insight into the requirements for a demanding six-electron, seven-proton reaction that has so far eluded synthetic catalysts, this study provides strategies for designing highly functional multicofactor artificial enzymes.

Gross, Z.

J. Am. Chem. Soc. 2005, 127, 2883-2887, 10.1021/ja045372c

An extremely simple biomimetic oxidation system, consisting of mixing metal complexes of amphiphilic corroles with serum albumins, utilizes hydrogen peroxide for asymmetric sulfoxidation in up to 74% ee. The albumin-conjugated manganese corroles also display catalase-like activity, and mechanistic evidence points toward oxidant-coordinated manganese(III) as the prime reaction intermediate.

Roelfes, G.

Angew. Chem. Int. Ed. 2018, 57, 7785-7789, 10.1002/anie.201802946

An artificial heme enzyme was created through self‐assembly from hemin and the lactococcal multidrug resistance regulator (LmrR). The crystal structure shows the heme bound inside the hydrophobic pore of the protein, where it appears inaccessible for substrates. However, good catalytic activity and moderate enantioselectivity was observed in an abiological cyclopropanation reaction. We propose that the dynamic nature of the structure of the LmrR protein is key to the observed activity. This was supported by molecular dynamics simulations, which showed transient formation of opened conformations that allow the binding of substrates and the formation of pre‐catalytic structures.

Ward, T.R.

Cat. Sci. Technol. 2018, 8, 2294-2298, 10.1039/C8CY00646F

We report an artificial carbenoid transferase which combines a biotinylated dirhodium moiety within streptavidin scaffold.

Hartwig, J.F.

Science 2016, 354, 102-106, 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.

Ward, T.R.

Chem. Sci. 2016, 7, 673-677, 10.1039/c5sc03116h

Introduction of a biotinylated monophosphine palladium complex within streptavidin affords an enantioselective artificial Suzukiase. Site-directed mutagenesis allowed the optimization of the activity and the enantioselectivity of this artificial metalloenzyme. A variety of atropisomeric biaryls were produced in good yields and up to 90% ee.

Ward, T.R.

J. Am. Chem. Soc. 2016, 138, 5781-5784, 10.1021/jacs.6b02470

Enzymes typically depend on either NAD(P)H or FADH2 as hydride source for reduction purposes. In contrast, organometallic catalysts most often rely on isopropanol or formate to generate the reactive hydride moiety. Here we show that incorporation of a Cp*Ir cofactor possessing a biotin moiety and 4,7-dihydroxy-1,10-phenanthroline into streptavidin yields an NAD(P)H-dependent artificial transfer hydrogenase (ATHase). This ATHase (0.1 mol%) catalyzes imine reduction with 1 mM NADPH (2 mol%), which can be concurrently regenerated by a glucose dehydrogenase (GDH) using only 1.2 equiv of glucose. A four-enzyme cascade consisting of the ATHase, the GDH, a monoamine oxidase, and a catalase leads to the production of enantiopure amines.

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.

Ward, T.R.

J. Organomet. Chem. 2005, 690, 4488-4491, 10.1016/j.jorganchem.2005.02.001

Based on the incorporation of biotinylated organometallic catalyst precursors within (strept)avidin, we have developed artificial metalloenzymes for the oxidation of secondary alcohols using tert-butylhydroperoxide as oxidizing agent. In the presence of avidin as host protein, the biotinylated aminosulfonamide ruthenium piano stool complex 1 (0.4 mol%) catalyzes the oxidation of sec-phenethyl alcohol at room temperature within 90 h in over 90% yield. Gel electrophoretic analysis of the reaction mixture suggests that the host protein is not oxidatively degraded during catalysis.

Kamer, P.C.J.; Laan, W.

ChemCatChem 2013, 5, 1184-1191, 10.1002/cctc.201200671

The development of artificial copper enzymes from sterol carrier protein type 2 like domain (SCP‐2L) for the use in asymmetric catalysis was explored. For this purpose, proteins were modified with various nitrogen donor ligands. Maleimide‐containing ligands were found most suitable for selective cysteine bio‐conjugation. Fluorescence spectroscopy was used to confirm copper binding to an introduced phenanthroline ligand, which was introduced in two unique cysteine containing SCP‐2L mutants. Copper adducts of several modified SCP‐2L templates were applied in asymmetric Diels–Alder reactions. A clear influence of both the protein environment and the introduced ligand was found in the asymmetric Diels–Alder reaction between azachalcone and cyclopentadiene. A promising enantioselectivity of 25 % ee was obtained by using SCP‐2L V83C modified with phenanthroline–maleimide ligand. Good endo selectivity was observed for SCP‐2L modified with the dipicolylamine‐based nitrogen donor ligand. These artificial metalloenzymes provide a suitable starting point for the implementation of various available techniques to optimise the performance of this system.

Lombardi, A.; Nastri, F.

Int. J. Mol. Sci. 2018, 19, 2896, 10.3390/ijms19102896

Many efforts are continuously devoted to the construction of hybrid biomaterials for specific applications, by immobilizing enzymes on different types of surfaces and/or nanomaterials. In addition, advances in computational, molecular and structural biology have led to a variety of strategies for designing and engineering artificial enzymes with defined catalytic properties. Here, we report the conjugation of an artificial heme enzyme (MIMO) with lipoic acid (LA) as a building block for the development of gold-based biomaterials. We show that the artificial MIMO@LA can be successfully conjugated to gold nanoparticles or immobilized onto gold electrode surfaces, displaying quasi-reversible redox properties and peroxidase activity. The results of this work open interesting perspectives toward the development of new totally-synthetic catalytic biomaterials for application in biotechnology and biomedicine, expanding the range of the biomolecular component aside from traditional native enzymes.

Artero, V.

ChemPlusChem 2016, 81, 1083-1089, 10.1002/cplu.201600218

The insertion of cobaloxime catalysts in the heme‐binding pocket of heme oxygenase (HO) yields artificial hydrogenases active for H2 evolution in neutral aqueous solutions. These novel biohybrids have been purified and characterized by using UV/visible and EPR spectroscopy. These analyses revealed the presence of two distinct binding conformations, thereby providing the cobaloxime with hydrophobic and hydrophilic environments, respectively. Quantum chemical/molecular mechanical docking calculations found open and closed conformations of the binding pocket owing to mobile amino acid residues. HO‐based biohybrids incorporating a {Co(dmgH)2} (dmgH2=dimethylglyoxime) catalytic center displayed up to threefold increased turnover numbers with respect to the cobaloxime alone or to analogous sperm whale myoglobin adducts. This study thus provides a strong basis for further improvement of such biohybrids, using well‐designed modifications of the second and outer coordination spheres, through site‐directed mutagenesis of the host protein.

Jarvis, A.G.

ACS Sustainable Chem. Eng. 2018, 6, 15100-15107, 10.1021/acssuschemeng.8b03568

We report novel artificial metalloenzymes (ArMs), containing tris(pyridylmethyl)amine (TPA), for the atom economic oxidation of lignin β-O-4 model compounds, using hydrogen peroxide. The protein scaffold alters the selectivity of the reaction from a low yielding cleavage reaction when using the parent Fe-tpa complex to a high yielding benzylic alcohol oxidation when using the complex incorporated into a protein scaffold, SCP-2L A100C. Engineering the protein scaffold to incorporate glutamic acid was found to improve the ArM activity, showing that rational design of the protein environment using metal binding amino acids can be a first step toward improving the overall activity of an artificial metalloenzyme.

Ward, T.R.

Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 4683-4687, 10.1073/pnas.0409684102

Most physiological and biotechnological processes rely on molecular recognition between chiral (handed) molecules. Manmade homogeneous catalysts and enzymes offer complementary means for producing enantiopure (single-handed) compounds. As the subtle details that govern chiral discrimination are difficult to predict, improving the performance of such catalysts often relies on trial-and-error procedures. Homogeneous catalysts are optimized by chemical modification of the chiral environment around the metal center. Enzymes can be improved by modification of gene encoding the protein. Incorporation of a biotinylated organometallic catalyst into a host protein (avidin or streptavidin) affords versatile artificial metalloenzymes for the reduction of ketones by transfer hydrogenation. The boric acid·formate mixture was identified as a hydrogen source compatible with these artificial metalloenzymes. A combined chemo-genetic procedure allows us to optimize the activity and selectivity of these hybrid catalysts: up to 94% (R) enantiomeric excess for the reduction of p-methylacetophenone. These artificial metalloenzymes display features reminiscent of both homogeneous catalysts and enzymes.

Roelfes, G.

ChemCatChem 2020, 12, 3190-3194, 10.1002/cctc.202000245

The supramolecular approach is among the most convenient methodologies for creating artificial metalloenzymes (ArMs). Usually this approach involves the binding of a transition metal ion complex to a biomolecular scaffold via its ligand, which also modulates the catalytic properties of the metal ion. Herein, we report ArMs based on the proteins CgmR, RamR and QacR from the TetR family of multidrug resistance regulators (MDRs) and Cu2+ ions, assembled without the need of a ligand. These ArMs catalyze the enantioselective vinylogous Friedel-Crafts alkylation reaction with up to 75 % ee. Competition experiments with ethidium and rhodamine 6G confirm that the reactions occur in the chiral environment of the hydrophobic pocket. It is proposed that the Cu2+-substrate complex is bound via a combination of electrostatic and π-stacking interactions provided by the second coordination sphere. This approach constitutes a fast and straightforward way to assemble metalloenzymes and may facilitate future optimization of the protein scaffolds via mutagenesis or directed evolution approaches.

Ward, T.R.

Angew. Chem. Int. Ed. 2008, 47, 701-705, 10.1002/anie.200703159

Palladium in the active site: The incorporation of a biotinylated palladium diphosphine within streptavidin yielded an artificial metalloenzyme for the title reaction (see scheme). Chemogenetic optimization of the catalyst by the introduction of a spacer (red star) between biotin (green triangle) and palladium and saturation mutagenesis at position S112X afforded both R‐ and S‐selective artificial asymmetric allylic alkylases.

Ward, T.R.

J. Am. Chem. Soc. 2003, 125, 9030-9031, 10.1021/ja035545i

Homogeneous and enzymatic catalysis offer complementary means to generate enantiomerically pure compounds. Incorporation of achiral biotinylated rhodium−diphosphine complexes into (strept)avidin yields artificial metalloenzymes for the hydrogenation of N-protected dehydroamino acids. A chemogenetic optimization procedure allows one to produce (R)-acetamidoalanine with 96% enantioselectivity. These hybrid catalysts display features reminiscent both of enzymatic and of homogeneous systems.

Ward, T.R.

J. Am. Chem. Soc. 2004, 126, 14411-14418, 10.1021/ja0476718

We report on the generation of artificial metalloenzymes based on the noncovalent incorporation of biotinylated rhodium−diphosphine complexes in (strept)avidin as host proteins. A chemogenetic optimization procedure allows one to optimize the enantioselectivity for the reduction of acetamidoacrylic acid (up to 96% ee (R) in streptavidin S112G and up to 80% ee (S) in WT avidin). The association constant between a prototypical cationic biotinylated rhodium−diphosphine catalyst precursor and the host proteins was determined at neutral pH:  log Ka = 7.7 for avidin (pI = 10.4) and log Ka = 7.1 for streptavidin (pI = 6.4). It is shown that the optimal operating conditions for the enantioselective reduction are 5 bar at 30 °C with a 1% catalyst loading.

Kamer, P.C.J.

ChemBioChem 2010, 11, 1236-1239, 10.1002/cbic.201000159

Pinning phosphines on proteins: A method for the cysteine‐selective bioconjugation of phosphines has been developed. The photoactive yellow protein has been site‐selectively functionalized with phosphine ligands and phosphine transition metal complexes to afford artificial metalloenzymes that are active in palladium‐catalysed allylic nucleophilic substitution reactions.

Borovik, A.S.; Don Tilley, T.

J. Am. Chem. Soc. 2018, 140, 2739-2742, 10.1021/jacs.7b13052

Artificial metalloproteins (ArMs) containing Co4O4 cubane active sites were constructed via biotin–streptavidin technology. Stabilized by hydrogen bonds (H-bonds), terminal and cofacial CoIII–OH2 moieties are observed crystallographically in a series of immobilized cubane sites. Solution electrochemistry provided correlations of oxidation potential and pH. For variants containing Ser and Phe adjacent to the metallocofactor, 1e–/1H+ chemistry predominates until pH 8, above which the oxidation becomes pH-independent. Installation of Tyr proximal to the Co4O4 active site provided a single H-bond to one of a set of cofacial CoIII–OH2 groups. With this variant, multi-e–/multi-H+ chemistry is observed, along with a change in mechanism at pH 9.5 that is consistent with Tyr deprotonation. With structural similarities to both the oxygen-evolving complex of photosystem II (H-bonded Tyr) and to thin film water oxidation catalysts (Co4O4 core), these findings bridge synthetic and biological systems for water oxidation, highlighting the importance of secondary sphere interactions in mediating multi-e–/multi-H+ reactivity.

Borovik, A.S.; Hendrich, M.P.; Moënne-Loccoz, P.

J. Am. Chem. Soc. 2021, 143, 2384-2393, 10.1021/jacs.0c12564

Dinuclear iron centers with a bridging hydroxido or oxido ligand form active sites within a variety of metalloproteins. A key feature of these sites is the ability of the protein to control the structures around the Fe centers, which leads to entatic states that are essential for function. To simulate this controlled environment, artificial proteins have been engineered using biotin–streptavidin (Sav) technology in which Fe complexes from adjacent subunits can assemble to form [FeIII–(μ-OH)–FeIII] cores. The assembly process is promoted by the site-specific localization of the Fe complexes within a subunit through the designed mutation of a tyrosinate side chain to coordinate the Fe centers. An important outcome is that the Sav host can regulate the Fe···Fe separation, which is known to be important for function in natural metalloproteins. Spectroscopic and structural studies from X-ray diffraction methods revealed uncommonly long Fe···Fe separations that change by less than 0.3 Å upon the binding of additional bridging ligands. The structural constraints imposed by the protein host on the di-Fe cores are unique and create examples of active sites having entatic states within engineered artificial metalloproteins.

Ward, T.R.

J. Am. Chem. Soc. 2006, 128, 8320-8328, 10.1021/ja061580o

Incorporation of biotinylated racemic three-legged d6-piano stool complexes in streptavidin yields enantioselective transfer hydrogenation artificial metalloenzymes for the reduction of ketones. Having identified the most promising organometallic catalyst precursors in the presence of wild-type streptavidin, fine-tuning of the selectivity is achieved by saturation mutagenesis at position S112. This choice for the genetic optimization site is suggested by docking studies which reveal that this position lies closest to the biotinylated metal upon incorporation into streptavidin. For aromatic ketones, the reaction proceeds smoothly to afford the corresponding enantioenriched alcohols in up to 97% ee (R) or 70% (S). On the basis of these results, we suggest that the enantioselection is mostly dictated by CH/π interactions between the substrate and the η6-bound arene. However, these enantiodiscriminating interactions can be outweighed in the presence of cationic residues at position S112 to afford the opposite enantiomers of the product.

Ward, T.R.

Angew. Chem. Int. Ed. 2011, 50, 3026-3029, 10.1002/anie.201007820

Man‐made activity: Introduction of a biotinylated iridium piano stool complex within streptavidin affords an artificial imine reductase (see scheme). Saturation mutagenesis allowed optimization of the activity and the enantioselectivity of this metalloenzyme, and its X‐ray structure suggests that a nearby lysine residue acts as a proton source during the transfer hydrogenation.

McNaughton, B.R.; Rovis, T.

J. Am. Chem. Soc. 2019, 141, 4815-4819, 10.1021/jacs.9b01596

Reliable design of artificial metalloenzymes (ArMs) to access transformations not observed in nature remains a long-standing and important challenge. We report that a monomeric streptavidin (mSav) Rh(III) ArM permits asymmetric synthesis of α,β-unsaturated-δ-lactams via a tandem C–H activation and [4+2] annulation reaction. These products are readily derivatized to enantioenriched piperidines, the most common N-heterocycle found in FDA approved pharmaceuticals. Desired δ-lactams are achieved in yields as high as 99% and enantiomeric excess of 97% under aqueous conditions at room temperature. Embedding a Rh cyclopentadienyl (Cp*) catalyst in the active site of mSav results in improved stereocontrol and a 7-fold enhancement in reactivity relative to the isolated biotinylated Rh(III) cofactor. In addition, mSav-Rh outperforms its well-established tetrameric forms, displaying 11–33 times more reactivity.

Hartwig, J.F.

ACS Cent. Sci. 2017, 3, 302-308, 10.1021/acscentsci.6b00391

Enzymes catalyze organic transformations with exquisite levels of selectivity, including chemoselectivity, stereoselectivity, and substrate selectivity, but the types of reactions catalyzed by enzymes are more limited than those of chemical catalysts. Thus, the convergence of chemical catalysis and biocatalysis can enable enzymatic systems to catalyze abiological reactions with high selectivity. Recently, we disclosed artificial enzymes constructed from the apo form of heme proteins and iridium porphyrins that catalyze the insertion of carbenes into a C–H bond. We postulated that the same type of Ir(Me)-PIX enzymes could catalyze the cyclopropanation of a broad range of alkenes with control of multiple modes of selectivity. Here, we report the evolution of artificial enzymes that are highly active and highly stereoselective for the addition of carbenes to a wide range of alkenes. These enzymes catalyze the cyclopropanation of terminal and internal, activated and unactivated, electron-rich and electron-deficient, conjugated and nonconjugated alkenes. In particular, Ir(Me)-PIX enzymes derived from CYP119 catalyze highly enantio- and diastereoselective cyclopropanations of styrene with ±98% ee, >70:1 dr, >75% yield, and ∼10,000 turnovers (TON), as well as 1,2-disubstituted styrenes with up to 99% ee, 35:1 dr, and 54% yield. Moreover, Ir(Me)-PIX enzymes catalyze cyclopropanation of internal, unactivated alkenes with up to 99% stereoselectivity, 76% yield, and 1300 TON. They also catalyze cyclopropanation of natural products with diastereoselectivities that are complementary to those attained with standard transition metal catalysts. Finally, Ir(Me)-PIX P450 variants react with substrate selectivity that is reminiscent of natural enzymes; they react preferentially with less reactive internal alkenes in the presence of more reactive terminal alkenes. Together, the studies reveal the suitability of Ir-containing P450s to combine the broad reactivity and substrate scope of transition metal catalysts with the exquisite selectivity of enzymes, generating catalysts that enable reactions to occur with levels and modes of activity and selectivity previously unattainable with natural enzymes or transition metal complexes alone.