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.

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.

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.

Roelfes, G.

Isr. J. Chem. 2015, 55, 21-31, 10.1002/ijch.201400094

Artificial metalloenzymes have emerged as a promising new approach to asymmetric catalysis. In our group, we are exploring novel artificial metalloenzyme designs involving creation of a new active site in a protein or DNA scaffold that does not have an existing binding pocket. In this review, we give an overview of the developments in the two approaches to artificial metalloenzymes for asymmetric catalysis investigated in our group: creation of a novel active site on a peptide or protein dimer interface and using DNA as a scaffold for artificial metalloenzymes.

Ward, T.R.

J. Organomet. Chem. 2004, 689, 4868-4871, 10.1016/j.jorganchem.2004.09.032

We report on the phenomenon of protein-accelerated catalysis in the field of artificial metalloenzymes based on the non-covalent incorporation of biotinylated rhodium–diphosphine complexes in (strept)avidin as host proteins. By incrementally varying the [Rh(COD)(Biot-1)]+ vs. (strept)avidin ratio, we show that the enantiomeric excess of the produced acetamidoalanine decreases slowly. This suggests that the catalyst inside (strept)avidin is more active than the catalyst outside the host protein. Both avidin and streptavidin display protein-accelerated catalysis as the protein embedded catalyst display 12.0- and 3.0-fold acceleration over the background reaction with a catalyst devoid of protein. Thus, these artificial metalloenzymes display an increase both in activity and in selectivity for the reduction of acetamidoacrylic acid.

Onoda, A.

J. Inorg. Biochem. 2016, 158, 55-61, 10.1016/j.jinorgbio.2015.12.026

A hybrid biocatalyst containing a metal terpyridine (tpy) complex within a rigid β-barrel protein nitrobindin (NB) is constructed. A tpy ligand with a maleimide group, N-[2-([2,2′:6′,2′′-terpyridin]-4′-yloxy)ethyl]maleimide (1), was covalently linked to Cys96 inside the cavity of NB to prepare a conjugate NB–1. Binding of Cu2 +, Zn2 +, or Co2 + ion to the tpy ligand in NB–1 was confirmed by UV–vis spectroscopy and ESI–TOF MS measurements. Cu2 +-bound NB–1 is found to catalyze a Diels–Alder reaction between azachalcone and cyclopentadiene in 22% yield, which is higher than that of the Cu2 +–tpy complex without the NB matrix. The results suggest that the hydrophobic cavity close to the copper active site within the NB scaffold supports the binding of the two substrates, dienophile and diene, to promote the reaction.

Makhlynets, O.V.

J. Inorg. Biochem. 2020, 212, 111224, 10.1016/j.jinorgbio.2020.111224

Metalloproteins constitute nearly half of all proteins and catalyze some of the most complex chemical reactions. Recently, we reported a design of 4G-UFsc (Uno Ferro single chain), a single chain four-helical bundle with extraordinarily high (30 pM) affinity for zinc. We evaluated the contribution of different side chains to binding of Co(II), Ni(II), Zn(II) and Mn(II) using systematic mutagenesis of the amino acids that constitute the primary metal coordination and outer spheres. The binding affinity of proteins for metals was then measured using isothermal titration calorimetry. Our results show that both primary metal coordination environment and side chains in the outer sphere of UFsc are highly sensitive to even slight changes and can be adapted to binding different 3d metals, including hard-to-tightly bind metal ions such as Mn(II). The studies on the origins of tight metal binding will guide future metalloprotein design efforts.

Mahy, J.-P.

Eur. J. Biochem. 2002, 269, 470-480, 10.1046/j.0014-2956.2001.02670.x

An artificial peroxidase‐like hemoprotein has been obtained by associating a monoclonal antibody, 13G10, and its iron(III)–α,α,α,β‐meso‐tetrakis(ortho‐carboxyphenyl)porphyrin [Fe(ToCPP)] hapten. In this antibody, about two‐thirds of the porphyrin moiety is inserted in the binding site, its ortho‐COOH substituents being recognized by amino‐acids of the protein, and a carboxylic acid side chain of the protein acts as a general acid base catalyst in the heterolytic cleavage of the O–O bond of H2O2, but no amino‐acid residue is acting as an axial ligand of the iron. We here show that the iron of 13G10–Fe(ToCPP) is able to bind, like that of free Fe(ToCPP), two small ligands such as CN–, but only one imidazole ligand, in contrast to to the iron(III) of␣Fe(ToCPP) that binds two. This phenomenon is general for a series of monosubstituted imidazoles, the 2‐ and 4‐alkyl‐substituted imidazoles being the best ligands, in agreement with the hydrophobic character of the antibody binding site. Complexes of antibody 13G10 with less hindered iron(III)–tetraarylporphyrins bearing only one [Fe(MoCPP)] or two meso‐[ortho‐carboxyphenyl] substituents [Fe(DoCPP)] also bind only one imidazole. Finally, peroxidase activity studies show that imidazole inhibits the peroxidase activity of 13G10–Fe(ToCPP) whereas it increases that of 13G10–Fe(DoCPP). This could be interpreted by the binding of the imidazole ligand on the iron atom which probably occurs in the case of 13G10–Fe(ToCPP) on the less hindered face of the porphyrin, close to the catalytic COOH residue, whereas in the case of 13G10–Fe(DoCPP) it can occur on the other face of the porphyrin. The 13G10–Fe(DoCPP)–imidazole complex thus constitutes a nice artificial peroxidase‐like hemoprotein, with the axial imidazole ligand of the iron mimicking the proximal histidine of peroxidases and a COOH side chain of the antibody acting as a general acid‐base catalyst like the distal histidine of peroxidases does.

Gross, Z.

Cat. Sci. Technol. 2011, 1, 578, 10.1039/c1cy00046b

Oxidation of thioanisoles, catalyzed by chiral manganese(III) and iron(III) corroles, provides the corresponding sulfoxides in moderate chemical yields and low enantioselectivities. Biocatalysis by non-chiral albumin-associated manganese(III) corroles proceeds much better and allows for the enantioselective synthesis of the pharmacologically important R-modafinil, in 88% yield and 73% ee.

Pordea, A.

J. Inorg. Biochem. 2021, 220, 111446, 10.1016/j.jinorgbio.2021.111446

Artificial metalloenzymes result from the insertion of a catalytically active metal complex into a biological scaffold, generally a protein devoid of other catalytic functionalities. As such, their design requires efforts to engineer substrate binding, in addition to accommodating the artificial catalyst. Here we constructed and characterised artificial metalloenzymes using alcohol dehydrogenase as starting point, an enzyme which has both a cofactor and a substrate binding pocket. A docking approach was used to determine suitable positions for catalyst anchoring to single cysteine mutants, leading to an artificial metalloenzyme capable to reduce both natural cofactors and the hydrophobic 1-benzylnicotinamide mimic. Kinetic studies revealed that the new construct displayed a Michaelis-Menten behaviour with the native nicotinamide cofactors, which were suggested by docking to bind at a surface exposed site, different compared to their native binding position. On the other hand, the kinetic and docking data suggested that a typical enzyme behaviour was not observed with the hydrophobic 1-benzylnicotinamide mimic, with which binding events were plausible both inside and outside the protein. This work demonstrates an extended substrate scope of the artificial metalloenzymes and provides information about the binding sites of the nicotinamide substrates, which can be exploited to further engineer artificial metalloenzymes for cofactor regeneration.

Song, W.J.

J. Inorg. Biochem. 2021, 223, 111552, 10.1016/j.jinorgbio.2021.111552

A large fraction of metalloenzymes harbors multiple metal-centers that are electronically and/or functionally arranged within their proteinaceous environments. To explore the orchestration of inorganic and biochemical components and to develop bioinorganic catalysts and materials, we have described selected examples of artificial metalloproteins having multiple metallocofactors that were grouped depending on their initial protein scaffolds, the nature of introduced inorganic moieties, and the method used to propagate the number of metal ions within a protein. They demonstrated that diverse inorganic moieties can be selectively grafted and modulated in protein environments, providing a retrosynthetic bottom-up approach in the design of versatile and proficient biocatalysts and biomimetic model systems to explore fundamental questions in bioinorganic chemistry.

Reetz, M.T.

Isr. J. Chem. 2015, 55, 51-60, 10.1002/ijch.201400087

Transition metal catalysis in asymmetric transformations plays a pivotal role in modern synthetic organic chemistry, with these catalysts being tuned by systematic variation of the chiral ligand. More than three decades ago it was recognized that an alternative approach is possible, namely the anchoring of an achiral ligand/metal entity in an appropriate protein host, with formation of an artificial metalloenzyme (hybrid catalyst). However, this procedure delivers a single transition metal catalyst, with high enantioselectivity being a matter of chance. In view of this restriction, we proposed in 2001/2002 the concept of directed evolution of such hybrid catalysts. The most intensively studied system involves biotinylated phosphine/metal entities which are non‐covalently anchored to streptavidin. The present review summarizes progress in this intriguing area of research. It includes the assessment of the requirements of a given Darwinian system to be successful, and offers hints on how to achieve success in future studies.

Hayashi, T

Isr. J. Chem. 2015, 55, 76-84, 10.1002/ijch.201400123

Heme can be removed from a number of native hemoproteins, thus forming corresponding apoproteins, each of which provides a site for binding of a metal complex. In one example, myoglobin, an O2 storage protein, can be reconstituted with iron porphycene to dramatically enhance the O2 affinity. Although it is known that myoglobin has poor enzymatic activity, the insertion of iron corrole or iron porphycene into apomyoglobin increases its H2O2‐dependent peroxidase/peroxygenase activities. Furthermore, reconstitution with manganese porphycene promotes hydroxylation of an inert CH bond. It is also of interest to insert a non‐porphyrinoid complex into an apoprotein. A cavity of apocytochrome c has been found to bind a diiron carbonyl complex, serving as a functional model of diiron hydrogenase. Aponitrobindin has a rigid β‐barrel structure that provides an excellent cavity for covalently anchoring a metal complex. A rhodium complex embedded in the cavity of genetically modified nitrobindin has been found to promote stereoselective polymerization of phenylacetylene.

Ward, T.R.

J. Organomet. Chem. 2009, 694, 930-936, 10.1016/j.jorganchem.2008.11.023

Incorporation of achiral biotinylated manganese-salen complexes into streptavidin yields artificial metalloenzymes for aqueous sulfoxidation using hydrogen peroxide. Four biotinylated salen ligands were synthesized and their manganese complexes were tested in combination with several streptavidin mutants, yielding moderate conversions (up to 56%) and low enantioselectivities (maximum of 13% ee) for the sulfoxidation of thioanisole.

Mahy, J.-P.

Eur. J. Biochem. 2004, 271, 1277-1283, 10.1111/j.1432-1033.2004.04032.x

In order to estimate the size of the cavity remaining around the heme of the 3A3–microperoxidase 8 (MP8) hemoabzyme, the formation of 3A3–MP8–Fe(II)‐nitrosoalkane complexes upon oxidation of N‐monosubstituted hydroxylamines was examined. This constituted a new reaction for hemoabzymes and is the first example of fully characterized Fe(II)–metabolite complexes of antibody–porphyrin. Also, via a comparison of the reactions with N‐substituted hydroxylamines of various size and hydrophobicity, antibody 3A3 was confirmed to bring about a partial steric hindrance on the distal face of MP8. Subsequently, the influence of the antibody on the stereoselectivity of the S‐oxidation of sulfides was examined. Our results showed that MP8 alone and the antibody–MP8 complex catalyze the oxidation of thioanisole by H2O2 and tert‐butyl hydroperoxide, following a peroxidase‐like two‐step oxygen‐transfer mechanism involving a radical–cation intermediate. The best system, associating H2O2 as oxidant and 3A3–MP8 as a catalyst, in the presence of 5% tert‐butyl alcohol, led to the stereoselective S‐oxidation of thioanisole with a 45% enantiomeric excess in favour of the R isomer. This constitutes the highest enantiomeric excess reported to date for the oxidation of sulfides catalyzed by hemoabzymes.

Latour, J.-M.

J. Inorg. Biochem. 2021, 225, 111613, 10.1016/j.jinorgbio.2021.111613

Amines are ubiquitous in biology and pharmacy. As a consequence, introducing N functionalities in organic molecules is attracting strong continuous interest. The past decade has witnessed the emergence of very efficient and selective catalytic systems achieving this goal thanks to engineered hemoproteins. In this review, we examine how these enzymes have been engineered focusing rather on the rationale behind it than the methodology employed. These studies are put in perspective with respect to in vitro and in vivo nitrene transfer processes performed by cytochromes P450. An emphasis is put on mechanistic aspects which are confronted to current molecular knowledge of these reactions. Forthcoming developments are delineated.

Ménage, S.

Isr. J. Chem. 2015, 55, 61-75, 10.1002/ijch.201400110

The principle of enzyme mimics has been raised to its pinnacle by the design of hybrids made from inorganic complexes embedded into biomolecules. The present review focuses on the design of artificial metalloenzymes for oxidation reactions by oxygen transfer reactions, with a special focus on proteins anchoring inorganic complexes or metal ions via supramolecular interactions. Such reactions are of great interest for the organic synthesis of building blocks. In the first part, following an overview of the different design of artificial enzymes, the review presents contributions to the rational design of efficient hybrid biocatalysts via supramolecular host/guest approaches, based on the nature of the inorganic complex and the nature of the protein, with special attention to the substrate binding. In the second part, the original purpose of artificial metalloenzymes has been twisted to enable the observation of transient intermediates, to decipher metal‐based oxidation mechanisms. The host protein crystals have been used as crystalline molecular‐scale vessels, within which inorganic catalytic reactions have been followed, thanks to X‐ray crystallography. These hybrids should be an alternative to enzymes for sustainable chemistry.

Jarvis, A.G.

J. Inorg. Biochem. 2021, 215, 111317, 10.1016/j.jinorgbio.2020.111317

Palladium catalysed reactions are ubiquitous in synthetic organic chemistry in both organic solvents and aqueous buffers. The broad reactivity of palladium catalysis has drawn interest as a means to conduct orthogonal transformations in biological settings. Successful examples have been shown for protein modification, in vivo drug decaging and as palladium-protein biohybrid catalysts for selective catalysis. Biological media represents a challenging environment for palladium chemistry due to the presence of a multitude of chelators, catalyst poisons and a requirement for milder reaction conditions e.g. lower temperatures. This review looks to identify successful examples of palladium-catalysed reactions in the presence of proteins or cells and analyse solutions to help to overcome the challenges of working in biological systems.

Oohora, K.

J. Inorg. Biochem. 2019, 193, 42-51, 10.1016/j.jinorgbio.2019.01.001

Electron transfer (ET) events occurring within metalloprotein complexes are among the most important classes of reactions in biological systems. This report describes a photoinduced electron transfer between Zn porphyrin and Fe porphyrin within a supramolecular cytochrome b562 (Cyt b562) co-assembly or heterodimer with a well-defined rigid structure formed by a metalloporphyrin–heme pocket interaction and a hydrogen-bond network at the protein interface. The photoinduced charge separation (CS: kCS = 320–600 s−1) and subsequent charge recombination (CR: kCR = 580–930 s−1) were observed in both the Cyt b562 co-assembly and the heterodimer. In contrast, interestingly, no ET events were observed in a system comprised of a flexible and structurally-undefined co-assembly and heterodimers which lack the key hydrogen-bond interaction at the protein interface. Moreover, analysis of the kinetic constants of CS and CR of the heterodimer using the Marcus equation suggests that a single-step ET reaction occurs in the system. These findings provide strong support that the rigid hemoprotein-assembling system containing an appropriate hydrogen-bond network at the protein interface is essential for monitoring the ET reaction.

Kitagawa, S.; Ueno, T.

Isr. J. Chem. 2015, 55, 40-50, 10.1002/ijch.201400097

Construction of artificial metalloenzymes based on protein assemblies is a promising strategy for the development of new catalysts, because the three‐dimensional nanostructures of proteins with defined individual sizes can be used as molecular platforms that allow the arrangement of catalytic active centers on their surfaces. Protein needles/tubes/fibers are suitable for supporting various functional molecules, including metal complexes, synthetic molecules, metal nanoparticles, and enzymes with high densities and precise locations. Compared with bulk systems, the protein tube‐ and fiber‐based materials have higher activities for catalytic reactions and electron transfer, as well as enhanced functions when used in electronic devices. The natural and synthetic protein tubes and fibers are constructed by self‐assembly of monomer proteins or peptides. For more precise designs of arrangements of metal complexes, we have developed a new conceptual framework, based on the isolation of a robust needle structure from the cell‐puncturing domains of a bacteriophage. The artificial protein needle shows great promise for use in creating efficient catalytic systems by providing the means to arrange the locations of various metal complexes on the protein surface. In this account, we discuss the recent development of protein needle‐based metalloenzymes, and the future developments we are anticipating in this field.

Holland, P.L.

J. Inorg. Biochem. 2021, 219, 111430, 10.1016/j.jinorgbio.2021.111430

Artificial metalloenzymes (ArMs) consist of an unnatural metal or cofactor embedded in a protein scaffold, and are an excellent platform for applying the concepts of protein engineering to catalysis. In this Focused Review, we describe the application of ArMs as simple, tunable artificial models of the active sites of complex natural metalloenzymes for small-molecule activation. In this sense, ArMs expand the strategies of synthetic model chemistry to protein-based supporting ligands with potential for participation from the second coordination sphere. We focus specifically on ArMs that are structural, spectroscopic, and functional models of enzymes for activation of small molecules like CO, CO2, O2, N2, and NO, as well as production/consumption of H2. These ArMs give insight into the identities and roles of metalloenzyme structural features within and near the cofactor. We give examples of ArM work relevant to hydrogenases, acetyl-coenzyme A synthase, superoxide dismutase, heme oxygenases, nitric oxide reductase, methyl-coenzyme M reductase, copper-O2 enzymes, and nitrogenases.

Herrick, R.S.

J. Organomet. Chem. 2014, 751, 90-110, 10.1016/j.jorganchem.2013.07.004

Bioorganometallic chemistry is a rapidly growing subfield of organometallic chemistry. One important facet is the study of organometallic•protein complexes that contain a covalent bond between the protein and an organometallic prosthetic group. Structural elucidation of these complexes is being used with increasing frequency to determine exactly where metal binding takes place and to obtain accurate structural information. This review summarizes the structures in this field, highlighting how this information has driven the frontier of this research.

Anderson, J.L.R.

J. Inorg. Biochem. 2021, 217, 111370, 10.1016/j.jinorgbio.2021.111370

The design and construction of de novo enzymes offer potentially facile routes to exploiting powerful chemistries in robust, expressible and customisable protein frameworks, while providing insight into natural enzyme function. To this end, we have recently demonstrated extensive catalytic promiscuity in a heme-containing de novo protein, C45. The diverse transformations that C45 catalyses include substrate oxidation, dehalogenation and carbon‑carbon bond formation. Here we explore the substrate promiscuity of C45's peroxidase activity, screening the de novo enzyme against a panel of peroxidase and dehaloperoxidase substrates. Consistent with the function of natural peroxidases, C45 exhibits a broad spectrum of substrate activities with selectivity dictated primarily by the redox potential of the substrate, and by extension, the active oxidising species in peroxidase chemistry, compounds I and II. Though the comparison of these redox potentials provides a threshold for determining activity for a given substrate, substrate:protein interactions are also likely to play a significant role in determining electron transfer rates from substrate to heme, affecting the kinetic parameters of the enzyme. We also used biomolecular simulation to screen substrates against a computational model of C45 to identify potential interactions and binding sites. Several sites of interest in close proximity to the heme cofactor were discovered, providing insight into the catalytic workings of C45.