Design Of Artificial Metalloenzymes Research Articles

Real-time observation of a metal complex-driven reaction intermediate using a porous protein crystal and serial femtosecond crystallography

Determining short-lived intermediate structures in chemical reactions is challenging. Although ultrafast spectroscopic methods can detect the formation of transient intermediates, real-space structures cannot be determined directly from such studies. Time-resolved serial femtosecond crystallography (TR-SFX) has recently proven to be a powerful method for capturing molecular changes in proteins on femtosecond timescales. However, the methodology has been mostly applied to natural proteins/enzymes and limited to reactions promoted by synthetic molecules due to structure determination challenges. This work demonstrates the applicability of TR-SFX for investigations of chemical reaction mechanisms of synthetic metal complexes. We fix a light-induced CO-releasing Mn(CO)3 reaction center in porous hen egg white lysozyme (HEWL) microcrystals. By controlling light exposure and time, we capture the real-time formation of Mn-carbonyl intermediates during the CO release reaction. The asymmetric protein environment is found to influence the order of CO release. The experimentally-observed reaction path agrees with quantum mechanical calculations. Therefore, our demonstration offers a new approach to visualize atomic-level reactions of small molecules using TR-SFX with real-space structure determination. This advance holds the potential to facilitate design of artificial metalloenzymes with precise mechanisms, empowering design, control and development of innovative reactions.

A Specific Guide for Metalloenzyme Designers: Introduction and Evolution of Metal-Coordination Spheres Embedded in Protein Environments.

ConspectusMetalloproteins establish a comprehensive molecular space by combining inorganic cofactors with protein environments. The chemical interplay between a metal element and a protein matrix is remarkable yet elusive, as the chemical properties of metal ions do not directly translate into those of metalloenzymes when placed within a protein matrix. Instead, the biochemical context determines the metal-coordination geometries, reaction kinetics, and thermodynamic parameters, such as redox potential and Lewis acidity/basicity. The molecular design of novel metalloproteins, which involves varying the combination of the two major components, metal ion and protein, allows us to validate our current understanding of their chemical interactions, modulate protein structure and function toward specific directions, and create protein-based functional biomaterials and biocatalysts. Considering theoretically possible protein sequence combinations and several metal elements at several oxidation states, artificial metalloenzyme design is analogous to the drawing of new paintings with multiple colors and brush strokes.Our laboratory has developed minimalistic strategies to transform diverse proteins into artificial metalloproteins with novel architectures or catalytic functions. First, we analyzed the geometric parameters of native Zn-binding sites to introduce a tetrahedral zinc-binding motif to a non-α-helical protein scaffold that shows neither pre-existing affinity to any metal ion nor catalytic activity, such as the β-barrel outer membrane protein OmpF. We created a series of artificial Zn-binding OmpF variants that possess coordinatively unsaturated mononuclear Zn-ligation sites and exhibit hydrolytic activities with esters, β-lactams, and β-glycosides. The OmpF variants were further evolved to zinc-dependent glycosidases, addressing a central question in bioinorganic chemistry: can zinc ions directly associate with the hydrolytic cleavage of glycosidic bonds in sugars?To expand the scope of metalloprotein design, we also utilized a metal-chelating noncanonical amino acid, bipyridine-alanine, and constructed tunable dicopper-dependent oxidases and nickel/iridium-dependent photocatalytic cross-coupling enzymes. These advancements increased the complexity and precision of metalloenzyme design, revealing how protein matrices, in conjunction with metallocofactors in aqueous media, facilitate electron transfer and mediate multiple chemical events, such as proton transfer, substrate access, and product release, while suppressing adventitious reactions.Furthermore, we redesigned the secondary and tertiary metal-coordination spheres of manganese-dependent quercetin dioxygenase and zinc-dependent β-lactamase, respectively. Biochemical characterizations of the evolved enzymes revealed that metal ions harmoniously and selectively cooperate with protein environments such that seemingly unimportant residues play critical roles in tuning the electronic structures of the enzymes or determining the metal-dependent reactivity. Thus, our work establishes the fundamental basis of how metalloenzymes function via long-range intermolecular interactions and how we can control their structure and function in specific directions. Consequently, these novel metalloenzymes have provided possible snapshots of how they could have evolved under controlled chemical environments and potential prospects for how to design more sophisticated and efficient biocatalysts with accuracy at the molecular level.

Application of engineered myoglobins for biosynthesis of clofazimine by integration with chemical synthesis.

Significant efforts have been made in the design of artificial metalloenzymes. Myoglobin (Mb), an O2 carrier, has been engineered to exhibit different functions. Herein, we applied a series of engineered Mb mutants with peroxidase activity for biosynthesis of clofazimine (CFZ), a potential drug with a broad-spectrum antiviral activity, by integration with chemical synthesis. Two of those mutants, F43Y Mb and F43Y/T67R Mb, have been shown to efficiently catalyze the oxidative coupling of 2-N-(4-chlorophenyl) benzene-1,2-diamine (N-4-CPBDA) in the presence of H2O2, with 97% yields. The overall catalytic efficiency (kcat/Km) is 46-fold and 82-fold higher than that of WT Mb, respectively. By further combination of this reaction with chemical synthesis, the production of CFZ was accomplished with an isolated yield of 72%. These results showed that engineered Mbs containing the Tyr-heme cross-link (F43Y Mb and F43Y/T67R Mb) exhibit enhanced activity in the oxidative coupling reaction. This study also indicates that the combination of biocatalysis and chemical synthesis avoids the need for the separation of intermediate products, which offers a convenient approach for the total synthesis of the biological compound CFZ.

Why surface hydrophobicity promotes CO2 electroreduction: a case study of hydrophobic polymer N-heterocyclic carbenes.

We report the use of polymer N-heterocyclic carbenes (NHCs) to control the microenvironment surrounding metal nanocatalysts, thereby enhancing their catalytic performance in CO2 electroreduction. Three polymer NHC ligands were designed with different hydrophobicity: hydrophilic poly(ethylene oxide) (PEO-NHC), hydrophobic polystyrene (PS-NHC), and amphiphilic block copolymer (BCP) (PEO-b-PS-NHC). All three polymer NHCs exhibited enhanced reactivity of gold nanoparticles (AuNPs) during CO2 electroreduction by suppressing proton reduction. Notably, the incorporation of hydrophobic PS segments in both PS-NHC and PEO-b-PS-NHC led to a twofold increase in the partial current density for CO formation, as compared to the hydrophilic PEO-NHC. While polymer ligands did not hinder ion diffusion, their hydrophobicity altered the localized hydrogen bonding structures of water. This was confirmed experimentally and theoretically through attenuated total reflectance surface-enhanced infrared absorption spectroscopy and molecular dynamics simulation, demonstrating improved CO2 diffusion and subsequent reduction in the presence of hydrophobic polymers. Furthermore, NHCs exhibited reasonable stability under reductive conditions, preserving the structural integrity of AuNPs, unlike thiol-ended polymers. The combination of NHC binding motifs with hydrophobic polymers provides valuable insights into controlling the microenvironment of metal nanocatalysts, offering a bioinspired strategy for the design of artificial metalloenzymes.

Reactivity of Myoglobin Reconstituted with Cobalt Corrole toward Hydrogen Peroxide.

The protein matrix of natural metalloenzymes regulates the reactivity of metal complexes to establish unique catalysts. We describe the incorporation of a cobalt complex of corrole (CoCor), a trianionic porphyrinoid metal ligand, into an apo-form of myoglobin to provide a reconstituted protein (rMb(CoCor)). This protein was characterized by UV-vis, EPR, and mass spectroscopic measurements. The reaction of rMb(CoCor) with hydrogen peroxide promotes an irreversible oxidation of the CoCor cofactor, whereas the same reaction in the presence of a phenol derivative yields the cation radical form of CoCor. Detailed kinetic investigations indicate the formation of a transient hydroperoxo complex of rMb(CoCor) which promotes the oxidation of the phenol derivatives. This mechanism is significantly different for native heme-dependent peroxidases, which generate a metal-oxo species as an active intermediate in a reaction with hydrogen peroxide. The present findings of unique reactivity will contribute to further design of artificial metalloenzymes.

Computationally driven design of an artificial metalloenzyme using supramolecular anchoring strategies of iridium complexes to alcohol dehydrogenase.

Artificial metalloenzymes (ArMs) confer non-biological reactivities to biomolecules, whilst taking advantage of the biomolecular architecture in terms of their selectivity and renewable origin. In particular, the design of ArMs by the supramolecular anchoring of metal catalysts to protein hosts provides flexible and easy to optimise systems. The use of cofactor dependent enzymes as hosts gives the advantage of both a (hydrophobic) binding site for the substrate and a cofactor pocket to accommodate the catalyst. Here, we present a computationally driven design approach of ArMs for the transfer hydrogenation reaction of cyclic imines, starting from the NADP+-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbADH). We tested and developed a molecular docking workflow to define and optimize iridium catalysts with high affinity for the cofactor binding site of TbADH. The workflow uses high throughput docking of compound libraries to identify key structural motifs for high affinity, followed by higher accuracy docking methods on smaller, focused ligand and catalyst libraries. Iridium sulfonamide catalysts were selected and synthesised, containing either a triol, a furane, or a carboxylic acid to provide the interaction with the cofactor binding pocket. IC50 values of the resulting complexes during TbADH-catalysed alcohol oxidation were determined by competition experiments and were between 4.410 mM and 0.052 mM, demonstrating the affinity of the iridium complexes for either the substrate or the cofactor binding pocket of TbADH. The catalytic activity of the free iridium complexes in solution showed a maximal turnover number (TON) of 90 for the reduction of salsolidine by the triol-functionalised iridium catalyst, whilst in the presence of TbADH, only the iridium catalyst with the triol anchoring functionality showed activity for the same reaction (TON of 36 after 24 h). The observation that the artificial metalloenzymes developed here lacked stereoselectivity demonstrates the need for the further investigation and optimisation of the ArM. Our results serve as a starting point for the design of robust artificial metalloenzymes, exploiting supramolecular anchoring to natural NAD(P)H binding pockets.

Design of artificial metalloenzymes with multiple inorganic elements: The more the merrier

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.

Design of artificial metalloenzymes for the reduction of nicotinamide cofactors

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. Synopsis about graphical abstractThe manuscript provides information on the design of artificial metalloenzymes based on the bioconjugation of rhodium complexes to alcohol dehydrogenase, to improve their ability to reduce hydrophobic substrates. The graphical abstract presents different binding modes and results observed with native cofactors as substrates, compared to the hydrophobic benzylnicotinamide.

A Hydroxyquinoline-Based Unnatural Amino Acid for the Design of Novel Artificial Metalloenzymes.

We have examined the potential of the noncanonical amino acid (8‐hydroxyquinolin‐3‐yl)alanine (HQAla) for the design of artificial metalloenzymes. HQAla, a versatile chelator of late transition metals, was introduced into the lactococcal multidrug‐resistance regulator (LmrR) by stop codon suppression methodology. LmrR_HQAla was shown to complex efficiently with three different metal ions, CuII, ZnII and RhIII to form unique artificial metalloenzymes. The catalytic potential of the CuII‐bound LmrR_HQAla enzyme was shown through its ability to catalyse asymmetric Friedel‐Craft alkylation and water addition, whereas the ZnII‐coupled enzyme was shown to mimic natural Zn hydrolase activity.

Molecular Modeling for Artificial Metalloenzyme Design and Optimization.

Artificial metalloenzymes (ArMs) are obtained by inserting homogeneous catalysts into biological scaffolds and are among the most promising strategies in the quest for new-to-nature biocatalysts. The quality of their design strongly depends on how three partners interact: the biological host, the "artificial cofactor," and the substrate. However, structural characterization of functional artificial metalloenzymes by X-ray or NMR is often partial, elusive, or absent. How the cofactor binds to the protein, how the receptor reorganizes upon the binding of the cofactor and the substrate, and which are the binding mode(s) of the substrate for the reaction to proceed are key questions that are frequently unresolved yet crucial for ArM design. Such questions may eventually be solved by molecular modeling but require a step change beyond the current state-of-the-art methodologies.Here, we summarize our efforts in the study of ArMs, presenting both the development of computational strategies and their application. We first focus on our integrative computational framework that incorporates a variety of methods such as protein-ligand docking, classical molecular dynamics (MD), and pure quantum mechanical (QM) methods, which, when properly combined, are able to depict questions that range from host-cofactor binding predictions to simulations of entire catalytic mechanisms. We also pay particular attention to the protein-ligand docking strategies that we have developed to accurately predict the binding of transition metal-containing molecules to proteins. While this aspect is fundamental to many bioinorganic fields beyond ArMs, it has been disregarded from the molecular modeling landscape until very recently.Next we describe how to apply this computational framework to particular ArMs including systems previously characterized experimentally as well as others where computation served to guide the design. We start with the prediction of the interactions between homogeneous catalysts and biological hosts. Protein-ligand docking is pivotal at that stage, but it needs to be combined with QM/MM or MD approaches when the binding of the cofactor implies significant conformational changes of the protein or involve changes of the electronic state of the metal.Then, we summarize molecular modeling studies aimed at identifying cofactor-substrate arrangements inside the ArM active pocket that are consistent with its reactivity. These calculations stand on "Theozyme"-like dockings, MD-refined or not, which provide molecular rationale of the catalytic profiles of the artificial systems.In the third section, we present case studies to decode the entire catalytic mechanism of two ArMs: (1) an iridium based asymmetric transfer hydrogenase obtained by insertion of Noyori's catalyst into streptavidin and (2) a metallohydrolase achieved by including a receptor. Transition states, second coordination sphere effects, as well as motions of the cofactors are identified as drivers of the enantiomeric profiles.Finally, we report computer-aided designs of ArMs to guide experiments toward chemical and mutational changes that improve their activity and/or enantioselective profiles and expand toward future directions.

Proteins as diverse, efficient, and evolvable scaffolds for artificial metalloenzymes.

By combining synthetic catalysts and biochemical tools, numerous artificial metalloenzymes have been designed to exhibit high catalytic activity and selectivity in diverse chemical transformations. Out of the nearly infinite number of discovered or characterised proteins, however, only a handful of proteins have been employed as scaffolds for artificial metalloenzymes, implying that specific proteins are preferred owing to their native structural, functional, or biochemical properties. In the present review, we extract and group the biochemical and structural properties of proteins that are advantageous in the design of artificial metalloenzymes; protein stability, pre-existing metal centre, native binding affinity for small molecules, confined and empty space, well-defined secondary structure, and native cellular location. The desirable properties highlight proteins as the key players in the design of metal-dependent biocatalysts. We also propose rarely considered, yet promising, proteins that could be versatile and unique scaffolds for novel metalloenzymes.

The Effect of Cofactor Binding on the Conformational Plasticity of the Biological Receptors in Artificial Metalloenzymes: The Case Study of LmrR.

The design of Artificial Metalloenzymes (ArMs), which result from the incorporation of organometallic cofactors into biological structures, has grown steadily in the last two decades and important new-to-Nature reactions have been reached. These type of exercises could greatly benefit from an understanding of the structural impact that the inclusion of organometallic moieties may have on the biological host. To date though, our understanding of this phenomenon is highly partial. This lack of knowledge is one of the elements that condition that first-generation ArMs generally display relatively poor catalytic profiles. In this work, we approach this matter by assessing the dynamics and stability of a series of ArMs resulting from the inclusion, via different anchoring strategies, of a variety of organometallic cofactors into the Lactococcal multidrug resistance regulator (LmrR) protein. To this aim, we coupled standard force field-based techniques such as Protein-Ligand Docking and Molecular Dynamics simulations with a variety of trajectory convergence analyses, capable of assessing both the stability and flexibility of the different systems under study upon the binding of cofactors. Together with the experimental evidence obtained in other studies, we provide an overview on how these changes can affect the catalytic outcomes obtained from the different ArMs. Fundamentally, our results show that the convergence analysis used in this work can assess how the inclusion of synthetic metallic cofactors in proteins can condition different structural modulations of their host. Those conformational modifications are key to the success of the desired catalytic activity and their proper identification can be wisely used to improve the quality and the rate of success of the ArMs.

Asymmetric δ-Lactam Synthesis with a Monomeric Streptavidin Artificial Metalloenzyme.

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.

Artificial Metalloenzyme Design with Unnatural Amino Acids and Non-Native Cofactors

There are 20 proteinogenic amino acids and a limited number of cofactors naturally available to build enzymes. Genetic codon expansion enables us to incorporate more than 200 unnatural amino acids into proteins using cell translation machinery, greatly expanding structures available to protein chemists. Such tools enable scientists to mimic the active site of an enzyme to tune enzymatic activity, anchor cofactors, and immobilize enzymes on electrode surfaces. Non-native cofactors can be incorporated into the protein through covalent or noncovalent interactions, expanding the reaction scope of existing enzymes. The review discusses strategies to incorporate unnatural amino acids and non-native cofactors and their applications in tuning and expanding enzymatic activities of artificial metalloenzymes.

Peroxidase Activity of a c-Type Cytochrome b5 in the Non-Native State is Comparable to that of Native Peroxidases.

The design of artificial metalloenzymes has achieved tremendous progress, although few designs can achieve catalytic performances comparable to that of native enzymes. Moreover, the structure and function of artificial metalloenzymes in non‐native states has rarely been explored. Herein, we found that a c‐type cytochrome b 5 (Cyt b 5), N57C/S71C Cyt b 5, with heme covalently attached to the protein matrix through two Cys–heme linkages, adopts a non‐native state with an open heme site after guanidine hydrochloride (Gdn⋅HCl)‐induced unfolding, which facilitates H2O2 activation and substrate binding. Stopped‐flow kinetic studies further revealed that c‐type Cyt b 5 in the non‐native state exhibited impressive peroxidase activity comparable to that of native peroxidases, such as the most efficient horseradish peroxidase. This study presents an alternative approach to the design of functional artificial metalloenzymes by exploring enzymatic functions in non‐native states.

Multidrug resistance regulators (MDRs) as scaffolds for the design of artificial metalloenzymes.

The choice of protein scaffolds is an important element in the design of artificial metalloenzymes. Herein, we introduce Multidrug Resistance Regulators (MDRs) from the TetR family as a viable class of protein scaffolds for artificial metalloenzyme design. In vivo incorporation of the metal binding amino acid (2,2-bipyridin-5yl)alanine (BpyA) by stop codon suppression methods was used to create artificial metalloenzymes from three members of the TetR family of MDRs: QacR, CgmR and RamR. Excellent results were achieved with QacR Y123BpyA in the Cu2+ catalyzed enantioselective vinylogous Friedel-Crafts alkylation reaction with ee's up to 94% of the opposite enantiomer that was achieved with other mutants and the previously reported LmrR-based artificial metalloenzymes.

Piano-stool d6-rhodium(III) complexes of chelating pyridine-based ligands and their papain bioconjugates for the catalysis of transfer hydrogenation of aryl ketones in aqueous medium

Two half-sandwich d6-rhodium(III) complexes of the general formula [(η5-Cp*)Rh(N^N)Cl]Cl where N^N is a phenanthroline or a bispyridine methane derivative carrying a thiol-targeting maleimide or chloroacetamide function were synthesized and characterized. Both complexes were able to catalyse the transfer hydrogenation of 2,2,2-trifluoroacetophenone in aqueous medium using formate or phosphite as hydrogen donor. Covalent anchoring of these complexes to the cysteine endoproteinase papain yielded hybrid metalloproteins with transfer hydrogenase properties. Under optimized conditions of pH, hydrogen donor concentration and catalyst load, conversion of substrate was nearly quantitative within 24h at 40°C and the (S)-enantiomer was obtained preferably albeit with a modest enantiomeric excess of 7⿿10%. Covalent docking simulations complemented the experimental findings suggesting a molecular rationale for the observed low enantioselectivity. The harmonious use of experimental and theoretical approaches represents an unprecedented starting point for driving the rational design of artificial metalloenzymes built up from papain with higher catalytic efficiency.

Toward the Computational Design of Artificial Metalloenzymes: From Protein–Ligand Docking to Multiscale Approaches

The development of artificial enzymes aims at expanding the scope of biocatalysis. Over recent years, artificial metalloenzymes based on the insertion of homogeneous catalysts in biomolecules have received an increasing amount of attention. Rational or pseudorational design of these composites is a challenging task because of the complexity of the identification of efficient complementarities among the cofactor, the substrate, and the biological partner. Molecular modeling represents an interesting alternative to help in this task. However, little attention has been paid to this field so far. In this manuscript, we aim at reviewing our efforts in developing strategies efficient to computationally drive the design of artificial metalloenzymes. From protein–ligand dockings to multiscale approaches, we intend to demonstrate that modeling could be useful at the different steps of the design. This Perspective ultimately aims at providing computational chemists with illustration of the applications of their too...

Special Issue: Artificial Metalloenzymes

Now is the perfect time to work in bioinorganic chemistry due to the rise of nanotechnology at the interface of biology and materials science. Although, until recently, bioinorganic chemistry was used to reconstruct metal-binding sites of natural metalloproteins and to elucidate the mechanisms of their functions, the role played by bioinorganic chemistry has changed from fundamental understanding to providing manipulation tools consisting of proteins with metals. In particular, the design of artificial metalloenzymes has grown to an important topic in biology and chemistry due to their potential applications in nanoscience and biotechnology. Several technologies have been developed using combinations of chemical and biological processes and computational methods. One of the general methods used in this field involves the encapsulation of non-natural metal cofactors within protein scaffolds. Because this methodology provides a powerful tool for generating new catalysts that promote single-step catalytic reactions with high activity and selectivity, the range of applications has been extended in pursuit of additional sophisticated and complex enzymatic systems with protein assemblies as employed in nature for elaborate enzymatic reactions such as photosynthesis and nitrogen fixation. In this special issue, thus, we focus on research of artificial metalloenzymes to lead the next generation of bioinorganic chemistry and biomaterial sciences. The issue is organized into nine sections: 1) P450 designed for non-natural reactivity, 2) P450 reactions using decoy molecules, 3) artificial metalloenzymes by cofactor modification of heme proteins, 4) artificial non-heme iron enzymes, 5) directed evolution of artificial metalloenzymes, 6) hybrid nanoreactors by Virus-Like Particles, 7) protein needles designed for catalytic reactions, 8) de novo designed helix bundles with metal binding sites, and 9) asymmetric catalysis in protein and DNA scaffolds. Each part contains articles by experts in the relevant area. The contributors to all the sections of the issue are extremely well known in their areas of research and have made significant contributions to the research of artificial metalloenzymes. We believe that this issue, which has a different scope from previous reviews on bioinorganic chemistry, supramolecular chemistry, and biomineralization, will be of interest not only to specialists but also to readers who are not familiar with bioinorganic chemistry. A large number of people have helped to put this issue together, so that it ultimately provides an invaluable resource for all those working on bioinorganic and coordination chemistry of artificial metalloenzymes. I would especially like to thank all the contributors, who have all produced excellent manuscripts, and made improvements to this issue. Finally, I would like to thank Dr. Brian Johnson, Managing Editor at Wiley-VCH, who worked very hard to assemble this issue on time.1 Takafumi Ueno Tokyo Institute of Technology Guest Editor

Oxidation Catalysis by Rationally Designed Artificial Metalloenzymes

AbstractThe 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.