Chem Soc RevAmbika Bhagi-Damodaran Ambika Bhagi-Damodaran was born in Moradabad, India. She received...

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Design and engineering of artificialoxygen-activating metalloenzymes

Flavia Nastri,a Marco Chino,a Ornella Maglio,ab Ambika Bhagi-Damodaran,c Yi Lu*c

and Angela Lombardi*a

Many efforts are being made in the design and engineering of metalloenzymes with catalytic properties

fulfilling the needs of practical applications. Progress in this field has recently been accelerated by

advances in computational, molecular and structural biology. This review article focuses on the recent

examples of oxygen-activating metalloenzymes, developed through the strategies of de novo design,

miniaturization processes and protein redesign. Considerable progress in these diverse design

approaches has produced many metal-containing biocatalysts able to adopt the functions of native

enzymes or even novel functions beyond those found in Nature.

1. Introduction

Enzymes are known to catalyze a wide variety of challengingchemical and biological reactions with high efficiency andselectivity, and these traits highlight how far Nature has evolvedenzymes towards catalyst optimization. A primary example isselective activation of dioxygen, which is far from simple andstill represents a major challenge for chemists.1 Natural

enzymes manage this task in a very efficient way, by usingcofactors, such as transition metal ions, highly conjugatedorganic molecules, or both.2,3 Oxygen-activating metalloenzymespredominately employ iron or copper ions within their active sites.For example, efficient dioxygen activation and substrate hydroxyl-ation are carried out by the heme monoxygenase cytochromesP450 (CYPs), where heme iron is coordinated by a thiolate of theCys residue.4 A variant of P450, OleTJE, is able to performoxidative decarboxylation chemistry, through hydrogen peroxideactivation.5,6 A wide range of reactions involving dioxygen arealso catalyzed by non-heme iron enzymes. Those enzymesinclude mononuclear non-heme iron oxygenases containing a2-His-1-carboxylate facial triad motif, such as the intradiol-cleaving catechol dioxygenases,7,8 and carboxylate-bridged

a Department of Chemical Sciences, University of Naples ‘‘Federico II’’, Via Cintia,

80126 Naples, Italy. E-mail: [email protected] IBB, CNR, Via Mezzocannone 16, 80134 Naples, Italyc Department of Chemistry, University of Illinois at Urbana-Champaign, A322 CLSL,

600 South Mathews Avenue, Urbana, IL 61801, USA. E-mail: [email protected]

Flavia Nastri

Flavia Nastri is a professor ofinorganic and bioinorganic chemi-stry at the University ‘‘Federico II’’of Napoli. She graduated inPharmaceutical Chemistry in 1990at the University of Napoli, whereshe received her PhD in 1996 inChemical Sciences. She was apostdoctoral fellow in the group ofProfessor Vincenzo Pavone at thesame University. In 1994 she joinedProfessor Daniel Mansuy’s group atthe Universite Rene Descartes, Paris(France). Her research interests are

centered on peptide and protein chemistry, covering the fields ofsynthesis and structural and functional characterization ofbiologically active peptides and metalloprotein models.

Marco Chino

Marco Chino received his BS andMS degrees in Chemistry from theUniversity of Napoli ‘‘Federico II’’.In 2014, he received his PhD inChemical Sciences under thesupervision of Professor AngelaLombardi, after spending sixmonths in San Francisco in theDeGrado Lab. He is now a post-doctoral fellow in the artificialmetalloenzyme group at theDepartment of Chemical Sciencein Napoli. His research interestsinclude computational proteindesign, bioinorganic chemistry,and protein–protein interactions.

Received 18th December 2015

DOI: 10.1039/c5cs00923e

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diiron proteins, such as the soluble methane monoxygenases,which hydroxylate a variety of organic substrates,9–11 and thestearoyl-acyl carrier protein (ACP) D9-desaturase, which intro-duces a double bond into saturated fatty acids.12,13 In additionto iron, copper is also utilized in enzymes involved in O2

binding, activation and subsequent substrate oxidation.14–16

The O2-reactive centers can be mononuclear (type 2 copper),dinuclear (type 3 copper) or trinuclear (type 2 and type 3copper), with no direct correlation between the type of coppercenter and reactivity.14–16 The type 2 copper sites are found inenzymes such as Cu,Zn superoxide dismutase and galactoseoxidase. The simplest of the metalloproteins containing thetype 3 copper center are hemocyanin (Hc), tyrosinase (Tyr), andcatechol oxidase (CatO). The type 3 sites are also found in themulticopper oxidases (MCOs), which include fungal laccase (Lc),

ceruloplasmin and ascorbate oxidase.17 More recently it wasdiscovered that cellulose and chitin degrading polysaccharidemonooxygenase (PMO) enzymes use only a single Cu center forcatalysis.18–22 The selected examples, mentioned above, areintended to emphasize the relevance of metal centers in biologyfor the activation of dioxygen and subsequent oxidative chemistry.This fascinating chemistry has received growing interest over theyears, and continuous efforts are made in preparing models ofdifferent sizes and complexities, to mimic the structures andfunctions of the natural counterparts.

Extensive studies on natural proteins and small moleculesynthetic catalysts have formed the basis of our current under-standing of dioxygen activation at various metal centers.A comprehensive description of small molecule mimics isbeyond the scope of this review, and readers can refer to several

Ornella Maglio

Ornella Maglio graduated inIndustrial Chemistry, in 1989,at the University of Napoli‘‘Federico II’’, where she receivedher PhD in Chemical Sciences in1992. In 1998 she was a visitingfellow at the National Institute ofMedical Research of London, inthe research group of Dr AnnalisaPastore. Since 2002 she has beena Researcher at the Institute ofBiostructures and Bioimages ofthe CNR in Naples. Her currentresearch interests include the

design and structural characterization, by NMR, of metallo-protein models, and model systems based on metal-bindingpeptides, reproducing the active site of natural metalloproteins.

Ambika Bhagi-Damodaran

Ambika Bhagi-Damodaran wasborn in Moradabad, India. Shereceived her BSc (Hons.) from St.Stephen’s College, Delhi, in 2009,followed by an MSc in Chemistryfrom the Indian Institute ofTechnology, Delhi, in 2011.Ambika completed her PhD inChemical Biology at the Universityof Illinois, Urbana-Champaign in2016 under the guidance ofProfessor Yi Lu focusing on theuse of myoglobin-based enzymemodels to probe structure–function

relationships in heme-copper oxidases. Her research interests includeenzyme design, spectroscopic and kinetic studies of metalloenzymes,protein–protein interactions and drug-design.

Yi Lu

Yi Lu received his BS degree fromPeking University in 1986, and aPhD degree from the University ofCalifornia, Los Angeles in 1992under Professor Joan S. Valentine.After two years of postdoctoralresearch in Professor Harry B.Gray’s group at Caltech, hestarted his own independentcareer in the Department ofChemistry, UIUC, in 1994. He isnow a Jay and Ann SchenckProfessor of Chemistry and aHHMI Professor in the Depart-

ments of Chemistry, Biochemistry, Bioengineering and MaterialsScience and Engineering. His research interests include design andengineering of metalloenzymes, and their applications inbiocatalysis for small-molecule activation, renewable energygeneration, environmental monitoring and medical diagnostics.

Angela Lombardi

Angela Lombardi is a professor ofinorganic chemistry and proteindesign at the University ‘‘FedericoII’’ of Napoli. She received herPhD in Chemical Sciences fromthe University of Napoli in 1990working in the area of transitionmetal chemistry and peptidesynthesis. After a visiting fellow-ship at the NIH, in 1990, shespent a sabbatical in ProfessorWilliam F. DeGrado’s group, atthe DuPont-Merck PharmaceuticalCo in 1995 and at the University of

Pennsylvania in 1997. Her research interests include the de novodesign of peptide-based metalloprotein models, as tools forunderstanding the structural requirements for function in naturalsystems, and their application in biosensing, diagnostics and catalysis.

Review Article Chem Soc Rev


excellent reviews, which collect the main results in this area.23–33

It should be outlined that these systems, which mainly reproducethe metal-site structure of their biological counterpart, have beenand still represent a benchmark for understanding the structure–function relationship in metalloproteins. They have deeply contri-buted to unravelling the role of the metal ion in the catalyticpathway, thus allowing a better interpretation of the chemistrysupported by the natural systems. Despite the important progressmade, most synthetic models developed so far are not able toreproduce some critical features of biocatalysts, such as high turn-over numbers under physiological conditions and high selectivity.The studies on small-molecule catalysts clearly demonstrated thatenzymatic catalysis requires not only information encoded inthe metal–ligand complex, but also those embodied within theprotein framework (e.g. the secondary coordination sphere),34

which is difficult to reproduce in small-molecules.The desire to develop artificial metalloenzymes that match

or even exceed the performance of natural enzymes has stimu-lated researchers toward the construction of functional metalsites into more elaborate architectures. Several strategies havebeen employed to construct peptide/protein-based artificialmetalloenzymes.35–47 In particular, three different design appro-aches, namely miniaturization processes,36,37 de novo design,35,38–41

and protein redesign,38,40–47 have been extensively and successfullyused to develop metalloenzyme mimics, and will be discussedin this review. The increasing number of structural data now-adays available, together with advanced computational methodsfor structure and sequence prediction, allows for the design ofuniquely packed structures and metal-binding sites within eitherde novo or native protein scaffolds. Computational algorithmshave been developed for designing metal-binding sites, bysearching protein structures for backbone geometries suitable toaccommodate the amino acid ligands in the appropriate con-formation for metal binding.48–52

Before examining specific cases, we wish to outline thecritical issues to be addressed and optimized simultaneously,in designing and engineering functional metalloenzymes:(1) the correct fold of the protein scaffold; (2) the coordinationrequirements of the metal ion; (3) the stability/activity trade-off,which is essential for the structure and function. First, theinteractions that contribute to protein folding should be preservedwhen designing a metal-binding site into a protein scaffold,mainly hydrophobic interactions that provide a powerful drivingforce for folding, polar interactions that are essential for conforma-tional specificity, and H-bonds that stabilize secondary and tertiarystructures.41,53,54 Secondly, for the construction of the metal-binding site, an appropriate number of coordinating amino acidresidues should be placed around the metal center, in the appro-priate position and conformation, in order to satisfy the coordina-tion geometry dictated by enzyme activity. More importantly,secondary coordination interactions, which play an important rolein positioning the ligand in the correct orientation for binding andin modulating the properties of the metal center, need to becarefully considered.34 Finally, a successful design of a metallo-enzyme requires a critical balance between conformational stabilityand catalytic activity of the enzyme. Structural metal-binding sites

in proteins frequently achieve stability by binding metal ions incoordinately saturated ligand environments, with idealized ligand–metal bond geometries.55,56 On the other hand, catalytic centersrequire coordinately unsaturated metal ions, sometimes withunusual geometries, to properly position and bind the substrates,to lower the activation energy for catalysis, and undergo confor-mational changes to perform catalysis.55,57 Thus, a successfulconstruction of a metalloenzyme reflects a delicate interplaybetween opposing requirements, that is tight binding of the metalcofactor versus function, as has been clearly highlighted throughthe analysis of artificial systems.57

Key lessons on how to construct functional metalloenzymescame not only from those involved in oxidation reactions, butalso in other activities. They represent major breakthroughs inprotein design and redesign, and are instructive for researchersin this field. Leading examples include artificial metallo-enzymes mimicking the catalytic activity of the natural enzymecarbonic anhydrase (CA), reported by Pecoraro and coworkers.58,59

Structurally much simpler than natural hydrolases, the artificialenzymes consist in de novo designed three-stranded coiled-coils.60,61 The most active model comprises a HgII ion forstructural stability, and an active site ZnII ion and catalyzesCO2 hydration with an efficiency comparable to some naturallyoccurring CAs.58 These studies proved that a functional site canbe engineered into a complete different scaffold, with respect tothe native enzyme. The designed coiled coils demonstrated wellsuited to accommodate the threefold symmetric histidine bindingsite of CA, thus allowing the authors to reproduce the coreelements necessary for function.

Computational and directed evolution methods also representkey tools in protein design: their combination has been proven tobe a valuable strategy for developing functional metalloenzymesand offers a general approach to explore new reactivities.Milestone results were reported by Baker and coworkers.62 Theyused RosettaMatch and RosettaDesign methodologies50,51 toidentify a set of mutations in adenosine deaminase that allowedit to catalyze the hydrolysis of a model organophosphate, by theinsertion of a zinc-binding site. The selected scaffold was furtherimproved by directed evolution, affording a redesigned enzymewith a catalytic efficiency (kcat/KM) of B104 M�1 s�1. The crystalstructure of the enzyme confirmed that all but one of the designedresidues adopt the designed conformation. These results show thepower of the approach, which can be readily applied to thegeneration of biocatalysts for different reactions. They highlightthe impact of joining computational design with directed evolution,in order to optimize the desired activities, by introducing keyelements, missed in the first stage.

Engineering desired functionalities into native protein scaffoldsoffers the advantage of bypassing the design of a stable proteinstructure. Lu and coworkers demonstrated that the nativemyoglobin (Mb) scaffold is well suited for the construction ofadditional metal-binding sites.63–65 The engineering of a non-heme iron binding center, through the incorporation of threehistidines and one glutamate in the distal pocket of myoglobin,afforded a nitric oxide reductase (NOR) mimic.63–65 This NORmimic not only reproduced the heme–non-heme diiron center

Chem Soc Rev Review Article


of NORs structurally but also functionally, by selectively reducingnitric oxide to nitrous oxide as in NORs.

All of the above examples demonstrate tremendous successachieved in protein design as a tool for understanding andmimicking native metalloenzymes. Success is measured notonly by how closely the designed metalloenzymes resemble thenative protein, but also by the insights gained from the designand engineering process. Numerous excellent reviews recentlypublished report the milestones reached in the design offunctional metalloenzymes.35,39,44,66–81 The aim of this reviewis to discuss the current research in the development ofmetalloenzymes active in oxidation chemistry, namely oxygenases,peroxidases and oxidases. Dioxygen activation and its transforma-tion are fundamental for both chemistry and biology, as they playkey roles in designing new catalysts for oxygen reduction reactionin fuel cells with high efficiency and for functionalization oforganic substrates.1 The triplet ground state and the strong O–Obond of molecular oxygen make it kinetically inert and quite stabletowards reaction at room temperature. Metalloenzymes utilizeparamagnetic metal ions like copper and iron to bind, activateand cleave the O–O bond of dioxygen and generate reactivehigh-valent species capable of oxidizing various substrates.We will highlight the efforts in constructing catalysts able topromote selective oxidations, with high regio- and stereo-selectivity, using ‘‘clean’’ oxidants, such as dioxygen and itstwo proton, and two electron reduced form – hydrogen peroxide.In the process, we will point out the challenges that have beenfaced and the successes achieved, by analyzing the results fromthe following perspectives: (i) capability of breaking the dioxygenbond and/or reacting with hydrogen peroxide to form reactivespecies; (ii) regio-/chemo-selectivity of the catalyzed reactions;(iii) turnover frequency/turnover number of the designed catalysts;(iv) adaptability of the catalyst to be tuned to oxidize a differentsubstrate; and (v) potential of the catalysts to be used in vivo viawhole-cell biotransformation for industrial purposes. The artifi-cial enzymes herein examined have been grouped according tothe design strategy adopted, namely miniaturization, de novodesign, and protein redesign. Using selected examples, we willtry to highlight the potential of each design strategy in expandingthe repertoire of protein functionalities and to provide perspectivesfor future development.

2. Artificial oxygen-activatingmetalloenzymes designed throughminiaturization

Many natural proteins have their functional properties locatedin discrete domains inside a large protein framework.82 Inmany cases, relatively few residues within these large surfacesactually participate in the catalytic activities. The approach ofprotein design through miniaturization aims to reduce theprotein size to obtain small structured polypeptides or ‘mini-proteins’, still retaining the protein functions. The goal of aminiaturization process is to design the minimal peptidesequence that contains sufficient information for proper folding

and for an accurate reconstruction of the active-site structure.36,37

This approach holds the advantage that the designed systems aregenerally simple enough, and therefore can be easily synthesizedand characterized. Simultaneously, the polypeptide sequencesare of sufficient size and chemical diversity to accommodatemetal-binding centers, if any. An essential requirement for theminiaturization process is the structural knowledge of the parentsystem. More specifically, it is necessary to define: (i) the typesand number of constituents to be assembled; (ii) the structureto be reconstructed and (iii) the function to be reproduced.Metalloproteins, in particular, are well suited for miniaturization.The metal center represents a pivot point where spheres ofvariable diameters that circumscribe part of the protein can becentered.37 The larger the diameter of the sphere, the larger thenumber of constituents that must be included in the model.Fig. 1 highlights this concept on the azurin crystal structure.83

Several strategies can be conceived to obtain a miniaturizedmodel: for example, two or more parts pulled out from the entireprotein could spontaneously associate to give folded non-covalent self-assembled oligomers, or two or more parts couldbe covalently connected through properly designed linkers to givefolded monomers. Moreover, starting with the simplest molecularmodel, one can iteratively introduce further levels of complexity,as guided by the experimental results, thus allowing the selectionof the minimal set of components dictating the desired structure,and finally housing the required function.

One of the most studied families of oxidative enzymes thathas been the subject of different miniaturization approaches isthat of heme-containing enzymes.84–86 In peroxidases, catalasesand monooxygenases, such as CYPs,87 catalysis takes place atthe heme iron center through the formation of a high-valentiron–oxo intermediate, whose fate depends on the specificenvironment created by the surrounding protein matrix.

Fig. 1 The concept of the miniaturization process highlighted on theazurin crystal structure (PDB ID: 4azu). The metal center represents a pivotpoint where spheres of variable diameters that circumscribe part of theprotein can be centered. The pink sphere includes all the elements ofthe first coordination sphere; the cyan sphere includes the elements ofthe secondary coordination sphere. The structure was generated usingPyMol.335



A notable class of ‘‘mini-hemeproteins’’ with catalytic activityare the microperoxidases (MPs), derived from the proteolyticdigestion of cytochrome c.88–91 The framework of MPs consistsof a small peptide fragment (generally containing from 8 to 11residues), containing the Cys–(Xaa)2–Cys–His motif, covalentlylinked to the heme c through thioether linkages with the twoCys residues.

The amino acid sequence of the polypeptide chain in MPs isnumbered according to that of the parent cytochrome c, i.e. thetwo Cys residues occupy positions 14 and 17 of the peptidechain. Selective proteolysis of cytochrome c affords variousMPs, differing in the peptide chain length.89 The best knownamong MPs is MP8, which is obtained from the tryptic digestionof horse heart cytochrome c; it retains the amino acid residues14–21 of the starting protein. MP11 containing residues 11–21,and MP9 containing residues 14–22 have also been studiedextensively. These MPs contain the minimal requirements for aheme-protein mimic, by retaining a His residue at position 18,which coordinates the heme iron and acts as a proximal ligand.In addition, the sixth coordination site to the heme is open,occupied by a H2O molecule, which is readily displaced by anentering exogenous ligand.

Despite their extensive use, no full atomic-resolution structurefor any MP is available owing to the conformational flexibility ofthe peptide portion. Recently, a new MP analog, MP9cb562, wasobtained from the tryptic digestion of cytochrome cb562. Theimmobilization of MP9cb562 inside a protein lattice cage allowedthe first crystallographic structure determination for any memberof the MP family (Fig. 2).92 MP9cb562 is anchored firmly to theprotein surface through the coordination of His73 (coming fromthe protein cage) to the iron, affording a bis-His heme. Thestructure of MP9cb562 confirms that c-type linkages have littleeffect on the heme conformation and therefore may primarilyserve other functions. For example such a linkage may affect theorientation of the coordinating His imidazole with respect to theporphyrin ring, which in turn may contribute to heme electronicasymmetry, and modulate electronic coupling to the heme iron.92

One of the most interesting aspects of the MP chemistryis that, despite their small size, they are able to selectivelyoxidize a variety of organic substrates, including ABTS, anilines,naphthols, phenols.93–99 MP11 has been shown to oxidizesulfides enantioselectively with modest enantiomeric excess (ee)values (16–25%).98 However, the application of MPs as catalystsis limited by their low stability under catalytic conditions. Theaccessibility of the distal side causes the degradation of theporphyrin ring during catalysis, either by the direct action ofH2O2 or by intermolecular reactions with another active iron–oxospecies. Despite these limitations, MPs are still much more stablethan simple protoporphyrin systems, indicating that the presenceof the small peptide chain can play an important protective roleand make the catalyst more robust. To minimize porphyrindegradation and to improve the stability of MPs for practicalapplications, several approaches have been undertaken. Oneapproach deals with the encapsulation of these enzymes inmesoporous metal–organic frameworks (MOFs).100–102 In adifferent approach, Casella and coworkers investigated the

effect of distal residues on the formation of the active high-valent iron–oxo intermediate upon reaction with H2O2.90,91

Several MP8 derivatives were synthesized by covalent linkageto the N-terminal amino group of one (Pro-MP8) or two (Pro2-MP8)proline residues, or a single proline N-protected by an aromaticfluorenyl group (Fmoc-Pro-MP8).99 The proline-modified MP8showed a faster rate of oxidation of p-cresol with hydrogen peroxiderelative to MP8. Further studies revealed that the reactivity of MP8derivatives is controlled by the polarity around the heme iron. InFmoc-Pro-MP8, the amine protecting group reduces the polarityand introduces some steric hindrance and as a consequence thecatalytic rate constant drops down with respect to the valueobserved for MP8 (k1 = 390 M�1 s�1 and k1 = 680 M�1 s�1, forFmoc-Pro-MP8 and MP8, respectively). In Pro-MP8, the protonatedproline –NH2

+ group acts as an internal acid–base catalyst in theheterolytic cleavage of the heme-bound peroxide, thus increasingthe activity of the catalyst (k1 = 1100 M�1 s�1). Moreover, theaddition of a second Pro residue further reduces the distancebetween the proline N atom and the peroxide oxygen atom,increasing the k1 value for Pro2-MP8 (k1 = 2000 M�1 s�1).99

Minimal heme-proteins able to promote oxidation reac-tion (named mimochromes) have been successfully obtainedby Lombardi, Pavone and coworkers through miniaturi-zation.84,85,103–113 This approach allowed designing the shortestpeptide sequences that could serve the same functions of thepeptide chain in the natural proteins (Fig. 3a and b). A detailedanalysis of heme-protein structures revealed that the prostheticgroup in natural heme-proteins is strongly kept inside theprotein structure by a large number of interactions, which werereplaced in mimochromes by a few strong local constraints.The prototype molecule, mimochrome I, was patterned on theF helix of the hemoglobin b-chain. Fig. 3c shows the X-raystructure of the b-chain Leu88–Leu96 F helix fragment facingthe heme group in human deoxyhemoglobin.114 Simple molecularmodeling studies showed that a change of conformation of theheme propionyl group and of the Lys95 sidechain from a foldedstate to an extended state was sufficient to bring the propionylcarboxyl moiety and the lysine b-amino functional group withinthe bonding distance (Fig. 3d). This covalent bond appeared as a

Fig. 2 Crystal structure of MP9cb562, obtained from the tryptic digestionof cytochrome cb562, and the distal position is occupied by the solventexposed His 73 from the co-crystallized cytochrome cb562 (PDB ID: 3m4c).



minimum requirement for positioning the helical scaffold inclose proximity to the heme iron center and to drive the peptidechain to cover the heme face upon His axial coordination.N-terminal acetylation and C-terminal amidation were then addedto avoid the presence of end charges that might affect the helixstability. Residues Ser89, Glu90, Cys93 and Asp94 were replacedwith Ala, Gln, Ala and Asn, respectively, in order to simplify thesynthetic procedure (Fig. 3e). The resulting sequence appeared tohave a high propensity to assume the desired helical folding, due tothe presence of five helix inducing residues (Leu1, 4, 9 and Ala2, 6)in a peptide of nine residues and of the N- and C-terminalprotections.115–117 Deuteroporphyrin was preferred as a cofactorover the more common protoporphyrin IX to avoid the degradationof the sensitive vinyl substituents during the synthesis. Finally, thehelix–heme–helix sandwich mimochrome I, shown in Fig. 3f, wasobtained by applying a C2 symmetry operation.103

The spectroscopic and structural features of mimochrome Iindicated that it binds cobalt and iron in a low-spin bis-His-ligation, as envisioned in the design, even though some unexpectedfeatures were observed.103–105 The iron complex showed quitelow solubility in water (in the mM range), thus limiting itspossible application as a functional heme-protein mimetic,whereas the cobalt complex was highly soluble (up to mMconcentration). This different behavior has been ascribed tothe stronger cobalt-histidine coordination that stabilizes thesandwich structure. Therefore, aggregation by stacking ofthe porphyrin ring occurs in the iron complex, causing a lowerwater solubility.

Starting from the mimochrome I prototype, numerouscompounds have been gradually produced in order to favorthe formation of a well-defined structure and to improve thewater solubility.106–109 In particular, mimochromes II and IVfeature a stable structure, with increased solubility in aqueoussolution (up to mM concentration), either as a free-base form,or as iron and cobalt complexes. To achieve this aim, twore-design strategies were used. The first strategy focused onpeptides with longer (14 residue) sequences, modeled in both

a-helical (residues 1–10) and extended (C-terminal part) con-formations, leading to mimochrome II.106 The second strategyconsisted of the introduction, into the mimochrome I sequence,of amino acid substitutions able to provide intra-molecular andinter-helical interactions, leading to mimochrome IV.107,108

Taking into account the lesson learned on the bis-His-ligatedcomplexes, attempts were made to stabilize a five-coordinateheme complex, apt to perform catalysis. A proximal and a distalsite environment of the heme were designed, such that: (i) atetradecapetide (TD) bearing a His residue at position 6 as theaxial ligand to the heme forms the proximal face and (ii) adecapeptide (D) devoid of any heme-coordinating residuecreates a cavity around the metal ion forms the distal face.As in mimochrome I, the two peptide chains embrace themetalloporphyrin in a fashion that the helical segments runantiparallel to each other and the helix axes are about parallelto the porphyrin plane. Stabilization of the tertiary structurewas contributed by inter-chain ion-pairs between the carboxy-late side chains of glutamate residues (Glu2) on one helix andthe guanidine groups of arginines (Arg10) on the other helix,strategy proven to be effective in mimochrome IV design.107

Moreover, the positively charged Arg10 and the negativelycharged Glu2 at the C-terminal and N-terminal ends, respectively(with opposite sign relative to the helix dipole), may also providestabilization to the secondary structure. Finally, several glutamines(Gln3, 4, 8) and a serine (Ser7) were introduced in the solventexposed positions to improve water solubility. The analoguemimochrome VI thus designed (Fig. 4) embodies some of thekey elements for functioning as a peroxidase-like catalyst.110,111

The spectroscopic and functional characterization indicatedthat it functions as an efficient heme-protein model, with aperoxidase-like catalytic activity. In the presence of hydrogenperoxide, it efficiently catalyzes the oxidation of several substratesexhibiting multiple turnovers. The mimochrome VI peptide frame-work, despite its small structure (a total of 24 amino acid residues),confers high efficiency to the porphyrin cofactor. Three importantoutcomes deserve highlighting: (i) FeIII–mimochrome VI efficiently

Fig. 3 Design through miniaturization: from hemoglobin (PDB ID: 2hhb) to mimochrome I. (a and b) Active site identification and extraction. (c) Isolationof heme covering the nonapeptide. (d) Mutual approach of heme propionate towards Lys92 by proper rotamer selection. (e) Selection of solvent exposedresidues and protoporphyrin IX to deuteroporphyrin mutation. (f) Symmetry-generated hexacoordinated deuteroheme. The structures were generatedusing PyMol.335



catalyzes the oxidation of different substrates, such as ABTS andguaiacol, by activating H2O2, and efficiently catalyzes the nitrationof phenols; (ii) FeIII–mimochrome VI displays a very high specificactivity (104 mol mg�1 s�1 for ABTS oxidation), with respect tohighly purified horse radish peroxidases (HRPs) (91 mol mg�1 s�1

for ABTS oxidation at pH 4.6); (iii) FeIII–mimochrome VI exhibitsmultiple turnover kinetics: more than 4000 turnovers wereobserved in the ABTS oxidation, without bleaching. The evidencethat both peptide chains modulate the properties of FeIII–mimochrome VI is a significant result, in terms of peroxidaseactivity. In fact, the derivative lacking the distal D chain showedhigher bleaching susceptibility, as indicated by the much lowerturnover number, suggesting a protective role of the D chain.

The success of the initial design demonstrates that it ispossible to incorporate a functional heme site within a minimalscaffold. Therefore, modification of the structure, throughspecific amino acid substitutions in the first and/or in thesecondary coordination shell, allowed for the structure–activityrelationship studies of mimochromes. To explore the role ofcharges in the peroxidase activity, Glu2 and Arg10 residueswere individually substituted in both the peptide chains withan uncharged residue.113 These substitutions were expected topartly destabilize the molecules due to the consequent removalof inter-chain and intra-chain interactions. To compensate thisstructure destabilization, leucine (Leu) was selected as thesubstituting residue, because of its high a-helical propensity,and for the potential hydrophobic interaction with the porphyrin.

As a result of these substitutions, four analogues were obtained:Glu2Leu(TD), Glu2Leu(D), Arg10Leu(TD), and Arg10Leu(D). Fig. 4reports the sequence modifications of the new analogues withrespect to mimochrome VI, by using single letter code for aminoacids, and the acronym MC6 for FeIII–mimochrome VI.

Table 1 lists the catalytic parameters for all the analogues.When compared to MC6 catalytic properties, kcat values areabout 2-fold higher in the analogues substituted at the (TD)chain. The best performances were obtained for E2L(TD)-MC6,which displays improvement in the apparent catalytic constant(kcat = 7.8 � 102 s�1), and efficiency for both H2O2 and ABTS(kcat/KM = 25 mM�1 s�1, and 16 � 103 mM�1 s�1, respectively).In contrast, the analogues with the altered D chain showed analmost unmodified reactivity, with respect to MC6. The E2L(D)-MC6 analogue was found to be the worst catalyst. Its kcat is3.8 � 102 s�1, and the kcat/KM values are 4.0 mM�1 s�1 for H2O2

and 3.3 � 103 mM�1 s�1 for ABTS.All MC6 analogues were able to perform several thousands

of turnovers, without degradation. The turnover numbers(TON) reflect the trend of the catalytic efficiency, being higherin E2L(TD)-MC6 and lower in R10L(TD)-MC6. Based on theE2L(TD)-MC6 model, it is possible to hypothesize a catalyticrole of R10 in the (D) chain. Its side chain may approach theferric ion and interact, by hydrogen-bonding, with the sixthligand to the heme. Hence, R10 is supposed to mimic R38 inthe HRP distal site and to enhance the efficiency of thecompound I formation and ligand binding.118 E2L(TD)-MC6behaves like natural peroxidases, whose reactivity is similarlyinfluenced by the proximal and distal heme environments.Moreover, the E2L(TD)-MC6 catalytic cycle occurs by a peroxidase-like mechanism, through the formation of compound I, containinga FeIVQO center and a porphyrin radical cation, as evidenced byspectroscopic analysis.

Collectively, the experimental results allowed pointing outthe principle in heme-protein reactivity: slight structural modi-fications in the heme-distal pocket may greatly influence theperoxidase-like activity. Moreover, the characterization of thisclass of minienzymes demonstrates that their miniaturizedstructure holds essential elements to finely tune the reactivity.This represents a very important checkpoint for the rationaldesign of new and improved bio-mimetic catalysts, tailored forspecific applications. The overall results demonstrate thatparticular attention in future design should be paid in order

Fig. 4 FeIII–mimochrome VI and its analogues. Left: Molecular model ofFeIII–mimochrome VI, highlighting the R10-E2 ion-pair interactions. Right:Peptide sequences of mimochrome VI and its analogues: acidic, basic andnon-polar residues, in positions 2 and 10, are indicated in red, blue andgreen, respectively. Reprinted with permission from ref. 113. Copyright2015 Royal Society of Chemistry.

Table 1 MC6 and its analogues’ steady-state kinetic parameters. HRP parameters are provided for comparisona

Enzyme pH

H2O2 ABTS

kcat � 10�2 (s�1)

H2O2 ABTS

TON � 10�3KM (mM) KM � 102 (mM) kcat/KM (mM�1 s�1) kcat/KM � 10�3 (mM�1 s�1)

MC6 6.5 44 � 2 8.4 � 0.2 3.7 � 0.1 8.4 � 0.6 4.4 � 0.2 4.0E2L(TD)-MC6 6.5 31 � 2 5.0 � 0.4 7.8 � 0.6 25 � 3 16 � 2 5.9R10L(TD)-MC6 6.5 54 � 2 3.8 � 0.1 6.8 � 0.3 13 � 1 18 � 1 5.6E2L(D)-MC6 6.5 96 � 7 11 � 1 3.8 � 0.3 4.0 � 0.6 3.4 � 0.6 3.6R10L(D)-MC6 6.5 18 � 1 3.0 � 0.2 1.7 � 0.1 9 � 1 5.7 � 0.7 3.3HRP 4.6 0.85 � 0.01 107 � 1 62 � 1 (7.3 � 0.2) � 103 5.8 � 0.1 50HRP 7.0 (1.15 � 0.01) � 10�2 (5.1 � 1.2) � 102 0.52 � 0.04 (4.6 � 0.5) � 103 (1.0 � 0.3) � 10�2 0.3

a Adapted from ref. 113.



to: (i) stabilize both the helical and the sandwiched structuresby introducing additional structural constraints; (ii) favor theHis-H2O coordination over a wider pH range; (iii) introduceamino acid residues assisting the acid–base catalytic cleavageof the O–O bond, as His42 acts in HRP.119

3. Artificial oxygen-activatingmetalloenzymes by de novo design

De novo metalloprotein design combines the fundamentalknowledge of protein design and biomimetic chemistry, andcan be defined as design ‘‘from first principles’’. The strategyinvolves the construction of a peptide sequence that is notdirectly related to any natural protein and is able to fold intoa unique, well-defined three-dimensional structure, and toincorporate a metal cofactor.39–41 De novo design is a valuableapproach to analyze the factors that tune the structural andfunctional properties of metal-binding sites in proteins. Thepossibility to isolate and investigate the active site of functionalmetalloproteins in a smaller, well-defined, model system allowsresearchers to accurately evaluate the role of first and secondaryshell interactions in modulating metalloprotein activity. Fig. 5illustrates the principal steps in the process of de novo proteindesign, applied by DeGrado, Lombardi and coworkers to thedevelopment of diiron oxo protein models, DFs.120–122 Thechoice of the structural motif to be reproduced is a critical firststep in the de novo design process (Fig. 5a). A simple structuralmotif, very common among natural, functionally diverse proteins,including many metalloproteins, is the four-helix bundle.53,123

This motif is compact, thermodynamically stable and able totolerate residue substitutions, deletions and insertions withoutperturbing the global folding. As a consequence, it representsa designable template in de novo design. The second step ofde novo design involves the generation of the backbone coordi-nates from scratch (Fig. 5b), in order to gain high qualitybackbone templates that are crucial for the success of thedesign process. Two main approaches are currently adopted

to generate backbone coordinates.119 One approach (referred toas ideal backbone parameterization) consists of modellingfolded structures through a few effective parameters, out ofthe very large number of parameters required to exactly definethe geometry of a protein.124 It has been demonstrated, forsome selected folds, that the approach can describe the structuralvariability, producing deviations between ideal and real structureswithin 1 Å. Examples of this include Crick parameterizationof coiled coils,124–126 mathematical description of b-barrelstructures,127 statistical parameterization of the structure ofcollagen,128 and parameterization of diiron helical bundles129,130

and of transmembrane helix interaction geometry.131 Thesecond approach consists of the fragment-based tertiary struc-ture composition. Fragmentation of protein structures reducesthe continuous protein structural space to a ‘‘parts list’’ ofpolypeptide building blocks.132 Since the pioneering study ofBaker and colleagues,133 several groups adopted peptide fragmentdatabases132 or atomistic tertiary fragment search,134–137 sub-stantially mining the Protein Database for robust and conservedbackbone arrangements apt to design.

The final step in de novo protein design (Fig. 5c) is thecareful identification of the sequences and their propertiesin order to stabilize the selected structure and to include thedesired function. This step requires the introduction andoptimization of a large number of short- and long-rangenon-covalent interactions, which all together contribute to thestability of a protein.41,53,54,138,139 This includes the correctplacement of hydrophobic and hydrophilic amino acids intothe sequence, H-bonds and electrostatic interactions and theintrinsic conformational propensity of each amino acid. Inaddition, for a de novo metalloprotein design, metal-bindingresidues need to be included, in the appropriate position andorientation for metal ion coordination (Fig. 5c). Finally, thesequence should also contain elements of negative design,which aim to stabilize the target fold and destabilize any otheralternative folds, thus providing conformational specificity. Forexample, inclusion of a large number of hydrophobic residuesin a designed protein can lead to multiple, stable conformations;

Fig. 5 Crucial steps in protein de novo design: from diiron proteins to DFs. (a) The four-helix bundle structural motifs have been identified as designabletemplates, due to their wide occurrence in natural diiron oxo proteins (bacterioferritin, PDB ID: 4am5, ribonucleotide reductase-R2, PDB ID: 1syy,p-aminobenzoate N-oxygenase, PDB ID: 3chh, and soluble methane monoxygenase hydroxylase, PDB ID: 1mty are reported). (b) Geometricalparameterization has been used to generate backbone coordinates from scratch. (c) The appropriate sequence has been selected, in order to stabilizethe desired tertiary structure, and to include the metal-binding residues. The DF1 structure and details of the diiron site are depicted (PDB ID: 1ec5).The structures were generated using PyMol.335



therefore, a correct balance between non-polar and polar inter-actions is required for correct folding and function.41,53,140,141

Examples of negative design include: (i) the placement ofhydrophilic groups that are solvent exposed in the desired foldbut buried in alternatively folded structures; (ii) inclusion ofburied H-bonds and salt bridges that are less stabilizing thanhydrophobic interactions, but can be formed only in the desiredstructure; (iii) solvent-accessible electrostatic interactions thatare weakly stabilizing the desired fold but strongly destabilizingthe alternatives. Other elements of negative design includeconformational restraints from residues such as Gly and Prothat are often used to interrupt elements of the secondarystructure. Shape complementarity and, more importantly, a lackthereof provide a third medium for negative design.

Because of the complexity of protein folding, an iterativeprocess is often necessary for de novo designing a metallo-protein with a native-like metal-binding structure. It has beendemonstrated that, through several cycles of design, synthesis,characterization and redesign, it is now possible to fine tunethe structural properties of the initial model, and to tailor thefunctional metal-site into the interior.122 Outstanding reportshave shown that a-helical bundles can be designed with thehighest degree of confidence. In particular, the ‘‘rules’’ thatcontrol stability, oligomerization, helix–helix orientation andmetal coordination are now well established and a variety ofde novo designed a-helical coiled coils and bundles, with native-like structures, have been reported.34,35,40,41,54 Since the firstexamples of designed proteins and metalloproteins,41 muchprogress has been made in the construction of functionalmetalloproteins, with efficiency approaching that of their naturalcounterparts. Some successful examples of de novo designedmetalloproteins with oxygen-activation activity are summarizedin the next paragraphs, with main focus on de novo design ofdiiron oxo proteins and heme-proteins.

3.1. Artificial oxygen-activating metalloenzymes by de novodesign of diiron oxo proteins

The ‘Due Ferri’ (two-iron; DF) family of proteins is a productof de novo protein design, successfully applied by DeGrado,Lombardi and coworkers for the development of artificialoxygen-activating metalloenzymes.120–122 The overall resultsobtained on the DF family demonstrated that the DF structurerepresents an excellent scaffold for hosting different activities.The earliest developed models have contributed to our under-standing of the principles governing protein folding, stabili-zation, as well as metal coordination and substrate binding.Through redesign processes, functional metalloproteins weresuccessfully produced. The progress in DF models, described inthe following paragraphs, clearly illustrates that, starting froma very stable de novo designed scaffold, a relatively smallnumber of amino acid mutations allowed shifting from inactiveto active proteins, or even to produce a remarkable switch ofactivity.

Although inspired by highly complex natural diironproteins,9,142–147 the original DF1 protein was designed usingmathematical parameterization of the backbone (Fig. 5).129,130

DF1 is an antiparallel dimer of the helix–loop–helix motif (a2),capable of binding the di-metal cofactor close to the center ofthe structure. The metal-binding site comprises four glutamatesand two histidines as first-shell ligands, which are positioned inthe core of the protein by hydrogen bonded interactions with twoaspartate, tyrosine and lysine residues.

Through an iterative process of design and characterization,catalytic diiron oxo sites were successfully engineered into theprototype DF1 structure. However, adding functionality in thisstable, uniquely folded DF1 protein required expansion of theactive site to accommodate substrates. Towards this aim,several DF1 analogues were designed, with changes in boththe sequence and loop conformation.148–155 The originalDF1 protein had a pair of Leu residues at positions 9 and 13(and 90 and 130 of neighboring helices), which entirely blockedaccess to the metal-binding site. By decreasing the bulk ofLeu13 with Ala or Gly, the formation of a cavity just above thedi-metal site was obtained, as indicated by crystal structureanalysis.151–153 However, to allow rapid and efficient substrateaccess and binding, it appeared that it would be necessary tosimultaneously mutate Leu9 to a smaller residue.

The sequence requirements for the catalytic activity werefirst evaluated in a combinatorial manner through the designof a four-chain four-helix bundle assembly composed of fourunconnected helical peptides (DFtet).

156–158 A series of asym-metrical variants were synthesized and mutations were performedat positions 15 and 19 (corresponding to the positions 9 and 13,respectively, of DF1). By mixing and matching different DFtet

peptides, it was possible to produce catalysts for the oxidation of4-amino-phenol (4-AP).158 The reaction involves the use of O2 tooxidize the diferrous protein to a diferric species (see Scheme 1).The diferric protein then reacts with the substrate 4-AP, producingbenzoquinone monoimine. The reduced diferrous form is thenoxidized by O2, thereby initiating another catalytic cycle. Thereleased quinone monoimine product is quenched and spectro-scopically detected by reacting with m-phenylen-diamine.159,160

The designated G4-DFtet analogue, in which both Leu15 andAla19 in two peptide chains were substituted with Gly,enhanced the reaction rate by E1000-fold relative to the back-ground reaction. G4-DFtet exhibited at least 100 turnovers with acatalytic efficiency of kcat/KM = 1540 M�1 min�1 (see Table 1).Mutating either of the Gly residues at positions 19 or 15 to Aladecreased the rate by approximately 2.5- to 5-fold.158 However,the complex stoichiometry, marginal stability (in the absence ofmetal ions), and tendency to undergo ligand-exchange reactions ofthese assemblies hampered any attempts to fully characterize theirthree-dimensional structure and catalytic properties.

Following this work, the authors sought to introduce aphenol-oxidase activity into the original DF1 helix–loop–helixscaffold, similar to the naturally occurring alternative oxidase(AOX) and plastid terminal oxidase (PTOX). These catalystscycle between di-FeII and di-FeIII states as they reduce O2, andthen use the oxidizing equivalents to convert quinols to quinones.161

To make an enzyme to reach an activity similar to PTOX, it wasessential to design a protein that is capable of: (1) generating a di-FeII

cofactor ready for dioxygen oxidation; (2) stabilizing the resulting



di-FeIII cofactor, which otherwise would be highly insoluble inaqueous solution; (3) binding phenols to allow two-electronoxidation of the substrate and regeneration of the diferrouscofactor; (4) releasing product, with concomitant entry into anew catalytic cycle. Furthermore, it was essential to engineerthe system so that each reaction occurs in a two-electronprocess, thereby avoiding Fenton chemistry, destruction ofthe catalyst, and accumulation of toxic radicals and by-products.Thus, it appeared that the design strategy should consider both thefunctional requirements already highlighted through the DFtet

protein subset as well as a careful redesign of the structuralframework. It was therefore necessary to redesign the loop tocompensate for stabilizing interactions lost during the incorpora-tion of four glycines into the hydrophobic core (a single mutationof Leu13 to Gly destabilized DF1 by 10.8 kcal mol�1 dimer�1).57 Toincrease the conformational stability of the DF scaffold, thesequence of the inter-helical turn was modified, affording thehelix–loop–helix dimer DF3 (Fig. 6a).162,163 This analogue inthe apo-form is only slightly less a-helical than the holo-form,but its thermodynamic stability outperforms DF1 mutants. DF3retained the catalytic activity already observed for G4-DFtet.In the presence of ambient oxygen, di-FeIII-DF3 followedMichaelis–Menten kinetics in the oxidation of 4AP, with valuesof 1.97 � 0.27 mM and 2.72 � 0.19 min�1, respectively, for KM

and kcat (Table 1, kcat/KM = 1380 M�1 min�1). Measurements ofthe reaction over the course of an hour indicated that theprotein was capable of at least 50 turnovers. As intended forthis particular design, the active site cleft in DF3 could accom-modate a larger substrate, such as 3,5-di-tert-butyl-catechol(3,5-DTBC). Di-FeIII-DF3 catalyzed the oxidation of this catecholderivative to the corresponding quinone (3,5-DTBQ), with a5-fold higher value of kcat/KM than that for 4-AP, reflecting anincrease in kcat (Table 2). Amino-aniline substrates were expectedto bind more weakly to the diferric center. Indeed, kcat/KM forthe two-electron oxidation of para-phenylenediamine (PPD) was75-fold lower than the value for 3,5-DTBC, and no catalysis wasobserved for ortho-phenylenediamine (OPD).

The encouraging results obtained with DFtet and DF3 analo-gues inspired the redesign of DFsc, the single-chain version ofDF1.164 The DFsc scaffold is made up of a single polypeptidechain, in which the four helices are linked by three computa-tionally designed loops. Four glycine residues were incorporatedalong the substrate access channel, affording the G4DFscvariant.165 Functional and spectroscopic analyses of this proteinprovide a detailed understanding of how solvent exposure andactive site ligation impact O2 reactivity. G4DFsc, as for the abovementioned members of the DF family with unencumberedsubstrate access channels, successfully catalyzed the two-electronoxidation of 4-aminophenol to the corresponding quinoneimmine. However, some drawbacks arose: substitutions of fourGly residues into the DFsc scaffold resulted in an apo-form slightlyless a-helical than the holo-form. In addition, better active siteaccessibility increases the exposure of the iron atoms to theaqueous solvent, which renders them prone to hydrolysis. Thisreduces the stability of the Fe–protein complex and leads to theprecipitation of iron oxides. Further spectroscopic analysesof G4DFsc showed that O2 binds to one iron and undergoes atwo-electron reduction to form an hemerythrin-like end-onhydroperoxo ferric intermediate. A larger access channel tothe active site induces solvent coordination at the diiron center,thus preventing O2 from binding to both iron ions to form abridged peroxy species, as observed in previous DFs. Thecoordinative saturation of the iron ions may account for the

Scheme 1 (a) Proposed mechanism for the oxidation of 4-AP. (b) Detectionof the produced quinone monoimine by reaction with m-phenylen-diamine.

Fig. 6 Catalytically active DF compounds. (a) Solution structure ofZn-DF3 (PDB ID: 2kik), and substrate conversion catalyzed by its ironderivative. (b) Solution structure of Zn-3His-DFsc (PDB ID: 2lfd), andsubstrate conversion catalyzed by its iron derivative. The structures weregenerated using PyMol.335

Table 2 Kinetics parameters obtained from the oxidation of the differentsubstratesa

Protein Substrate KM (mM) kcat (min�1)kcat/KM(M�1 min�1)

Di-Fe(III)-DF3 3,5-DTBC 2.09 � 0.31 13.2 63154-AP 1.97 � 0.27 2.72 1380PPD 8.87 � 2.58 0.73 83OPD Not detected

G4-DFtet 4-AP 0.83 � 0.06 1.30 1540




lack of oxidase activity of the diferric species. As a consequence,G4DFsc oxidase activity is observed when substrate binding tothe biferrous site preceded O2 binding (Scheme 2).166,167

The reactivity potential achieved in the DFsc scaffold hasbeen recently demonstrated by the switching of its activity fromphenol oxidase to N-hydroxylase, through the incorporation offour mutations at different levels: one first-shell, two secondaryshell and one third-shell.165 An additional iron-bindinghistidine was introduced in the active site to mimic the activesite of the natural diiron oxo protein p-aminobenzoateN-oxygenase (AurF).168 The steric clashes caused by the insertionof an additional ligand in the active site were overcome by theinsertion of computationally screened mutations. The resulting3His-G4DFsc (Fig. 6b) catalyst showed oxygenase activity,efficiently converting p-anisidine to the corresponding hydroxyl-amine, with the loss of hydroquinone oxidation activity understeady state conditions. Spectroscopic studies demonstrated thatthe substrate is bound in the active site, which is able to cyclebetween diferrous and ferric states, allowing for several turn-overs to take place (Scheme 3).166 Furthermore, the loss ofoxidase activity was attributed to the coordinative saturation ofthe metal center by the addition of the third histidine leadingto a very slow substrate binding kinetics, which may be ratelimiting.166

The overall work done on DF proteins highlights the powerin using de novo design for the development of simplifiedmetalloproteins as tools for future investigations on the geometricand electronic aspects that tune the diiron oxo site properties, andfor the design of novel catalysts with a wide variety of reactivities.

3.2. Artificial oxygen-activating metalloenzymes by de novodesign of heme-proteins

Efforts to develop heme-protein models by de novo design havegreatly increased over the years, due to the important rolesplayed by heme, which include dioxygen transport and storage,electron transfer, and dioxygen activation. The high level of

confidence reached in the design of structured four-helixbundles allowed the construction of several heme-proteinmimetics, in which the four-helix bundles serve as scaffoldsfor mono-heme and multi-heme binding.35 First and secondaryshell interactions were accurately introduced in order to accom-plish specific functions, i.e. electron transfer,169–172 oxygenbinding,173 hydroxylase,174 oxygenase,175 and peroxidase176–178

activities in de novo designed proteins.Several approaches have been undertaken to develop functional

heme-proteins by de novo design, and examples include thetemplate-assembled synthetic four-helix bundle proteins,175,179–183

and the construction of variable gene libraries.176–178,184–186

The first example of functional heme-protein mimetics,Helichrome, has been reported by Sasaki and Kaiser.174 Theyused the Template-Assembled Synthetic Protein (TASP) approach,introduced by Mutter and coworkers that consists of selectingan appropriate scaffold for the covalent assembly of a variety oftertiary structures.187–189 Helichrome represents a pioneeringexample of a catalytic heme-containing four-helix bundle, inwhich the porphyrin is also used as a template to generate aC4-symmetric four-helix bundle. This molecule is made up of fouridentical helical 15-residue peptide chains, N-terminally linked tothe four propionic groups of coproporphyrin I. Helichrome wasdesigned to mimic the hydroxylase activity of CYPs, whosestructure is characterized by the presence of several a-helicessurrounding the heme group forming a hydrophobic substraterecognition site. To this aim, the peptide sequence in Helichromewas chosen such that it could stabilize an amphiphilic helix andcreate, in the folded state, a substrate binding hydrophobicpocket distal to the porphyrin ring, as schematically depictedin Fig. 7. The FeIII complex, similar to CYPs, catalyzed thehydroxylation of aniline to form p-aminophenol, using O2 as anoxidant, in the presence of 7-acetylflavin and NADPH as sacri-ficial reductants, with kcat and KM values of 0.02 min�1 and5.0 mM, respectively. Several heme-proteins, such as hemoglobin,indoleamine 2,3-dioxygenase, and L-tryptophane 2,3-dioxygenase,

Scheme 2 Proposed mechanism for the oxidation of 4-AP by G4DFsc. Reprinted with permission from ref. 166. Copyright 2015 American ChemicalSociety.



have been reported to catalyze the hydroxylation of aniline in thepresence of oxygen and appropriate reducing agents with kcat andKM ranging from 0.02 to 0.65 min�1 and from 3.7 to 5.4 mM,respectively.174 Comparison of kcat and KM values clearly demon-strates that Helichrome has a hydroxylase activity quite similar tonative heme-proteins. Interestingly, the rigid hydrophobic pocketformed by the peptide chains plays a key role in Helichromeactivity (potentially by bringing the substrate in close proximity tothe heme iron) as the FeIII complex of the isolated coproporphyrinshowed negligible catalytic activity under similar conditions.

Following a similar approach, Haehnel and coworkersdeveloped Modular Organized Proteins, MOPs, as suitablescaffolds for the incorporation of a variety of functions.175,179–182

The first molecule capable of binding heme, MOP1, was

engineered using four a-helical peptides covalently connectedto a cyclic decapeptide template.179 Subsequently, a bis-heme-binding protein was constructed on the basis of the cytochromeb subunit of the mitochondrial cytochrome bc1 complex. Twohelices, H1 and H2, were designed in an attempt to reproducethe main features of the cytochrome b A–D helices: H1 con-stitutes the heme-binding helices and was covalently linked tothe template in a parallel orientation. They run antiparallel to aparallel H2 helix couple, which plays the role of shielding theheme-binding pocket against the solvent (Fig. 8a). The spectro-scopic properties of the bound heme resembled those of thenatural protein suggesting the successful assembly of themodel heme-protein. Further improvement in the design ofMOPs resulted in the development of artificial heme-proteinswith oxygenase activity, supported on cellulose membranes.175

To this aim, a library of mono-His-ligated heme-proteins wasprepared using the peptide sequences derived from MOP1. Asuitable chemoselective synthetic strategy was adopted in orderto control the directionality of the helical segments (Fig. 8b).Amino acid residues presumed to be in contact with the hemegroup were varied, in order to optimize heme binding andheme oxygenase activity. Heme oxygenase is an unusualenzyme in that the substrate heme serves also as a cofactor.It catalyzes the degradation of heme to verdoheme, which canbe followed in vitro in the presence of ascorbate as an electrondonor. A total of 352 synthetic heme-proteins were screened onthe cellulose support, by monitoring the kinetics of verdohemeformation, which showed first-order rate constant k1 valuesranging by more than two orders of magnitude, between0.1 � 10�3 and 50 � 10�3 min�1. Four out of 352 proteins wereselected to cover a broad range of enzymatic activities (k1 inthe range of 2.6 � 10�2 to 0.01 � 10�2 min�1) for full charac-terization in solution. Faster reaction rates k1 of the soluble as

Scheme 3 Proposed mechanism for the oxidation of p-anisidine by 3HisG4DFsc. Reprinted with permission from ref. 166. Copyright 2015 AmericanChemical Society.

Fig. 7 Proposed structure of Helichrome after folding of the peptidechains. Reprinted with permission from ref. 174. Copyright 1989 AmericanChemical Society.



compared to the immobilized proteins were observed. Moreover,two proteins showed reaction rates comparable to those ofmyoglobin mutants.175 Solution characterization revealed thatthe heme oxygenase activity was strictly related to the hemecoordination and redox potential. In conclusion, the TASPapproach was effective in the construction of stable four-helixbundles housing a functional heme, and found to be verypromising for engineering different scaffolds housing a varietyof metal-binding sites.

The screening of a library of variants was also used by Hechtand coworkers for the design of heme-binding proteins basedon the four-helix bundle scaffold.176–178,184–186 These authorsdeveloped a method for designing libraries of novel proteinsthat relies on the ‘‘binary patterning’’ of polar and non-polaramino acids.190,191 Binary patterning incorporates polar andnon-polar amino acids in accordance with the structural perio-dicity of the target secondary structure. This binary code doesnot require to explicitly specify the residue identity, thereforethe method has great potential for combinatorial diversity.192

Several libraries of sequences were generated and tested.Screening of hundred arbitrarily chosen sequences affordedmany sequences able to bind heme; the resulting proteins hadactivities ranging from electron transfer184,185 to carbon monoxidebinding.186 Furthermore, several designed proteins exhibitedperoxidase activity at rates rivaling natural peroxidases.176 Morerecently, the authors sought to mimic natural selection by intro-ducing random mutations to produce variants with improvedactivity.178 Directed molecular evolution on two previouslycharacterized proteins, S824 and S836, yielded novel mutatedsequences. The authors developed two different screens forperoxidase activity: a 96-well format that allowed for screeningseveral hundred sequences, and a colony-based assay thatallowed screening of thousands of clones. Several rounds of

screening and selection allowed the identification of the twoprogeny mutants that were nearly 3-fold more active than theparental sequence S836. The overall results demonstrate thatthe binary-patterned sequences, even though not related to anynatural protein and not specifically designed for any catalyticactivity, are useful starting materials for directed evolutiondesigns. The peculiarity of the overall approach lies on theapplication of evolution to novel sequences that can be specifiedtoward a desired biological function.

A further example of a de novo designed four-helix bundleheme-protein with peroxidase activity is MiniPeroxidase 3(Fig. 9).193 The design was inspired by the bacterioferritin andHRP structures. By covalently linking deuteroporphyrin to twopeptide chains of different compositions, an asymmetrica2/heme/a2 sandwich was obtained. The protein holds: (i) aHis residue on one chain that acts as an axial ligand to the ironion; (ii) a vacant distal site able to accommodate exogenous ligandsor substrates; and (iii) an Arg residue in the distal site that shouldassist hydrogen peroxide activation as in HRP. MiniPeroxidase 3was synthesized and characterized as its iron complex, and itsspectroscopic and catalytic properties were compared with those ofnatural systems. FeIII–MiniPeroxidase 3 shows high catalytic turn-over (kcat = 535 s�1) and efficiency (kcat/KM = 3.1 mM�1 s�1) in theoxidation of ABTS by H2O2. However, the observed activity is lowerwith respect to natural HRP, probably due to the mobility of thecatalytic Arg in the distal site.

In conclusion, all the examples discussed above demon-strate the successful use and implementation of the de novodesign strategy to obtain heme-enzymes with increased catalyticefficiency, turnover numbers, and robustness.

4. Artificial oxygen-activatingmetalloenzymes by protein redesign

As outlined in the previous section, de novo protein design hasbeen quite successful in developing artificial metalloenzymes.

Fig. 8 De novo designed MOPs. (a) Model of the MOP1 bis-hemecomplex. The backbone is shown as a ribbon. Amino acid side chainsare shown to illustrate the thioether linkage of the four Cys of the templatewith the N-terminal two Gly of helix H1 and the C-terminal Lys of helix H2.The His residues which bind the two heme groups and the Phe residuein the center of helix H1 are presented as stick structures. N and C indicatethe N- and C-terminus of helices H2 and H1, respectively. Reprinted withpermission from ref. 179, Copyright 1998, American Chemical Society.(b) Modular protein synthesis on peptide-templates linked to cellulose. Thefour-helix bundle protein consists of three different helix types, Ai (usedtwice in the assembly), Bj, and Ck, of which helix Bj provides the His residueas a ligand to the heme. Reprinted with permission from ref. 175, Copyright2007, Elsevier.

Fig. 9 Computer model of MiniPeroxidase 3. Trace representation of thehelices with key residues depicted as sticks: the His32-coordinating residueand the hydrogen-bonded Asp9 residue on the proximal site, the catalyticArg13 residue on the distal site, and the heme group, which is covalentlylinked to the peptide chains through the side chain of two lysine residues.Adapted with permission from ref. 193, Copyright 2012 Wiley.



Despite the success, this approach is limited by the types ofde novo designed scaffolds (mainly based on a-helical bundles),and thus by the range of protein environments available forcontrolling the activity and the selectivity of the metal-bindingsites. Since the types of native protein scaffolds are much morethan de novo designed scaffolds, designing artificial enzymesusing native scaffolds can compensate for this particular limi-tation of the de novo design approach, but a careful choice ofthe scaffold must be performed.38,42–44 Fortunately, there areseveral natural protein scaffolds available with high stabilityand therefore much more tolerant to mutations for the con-struction of a metal center, making it more likely for industrialapplications. Nature is a master at taking a stable scaffold thathas been evolved for millions of years and engineering differentactive sites into the same scaffold for different functions.Learning this ‘‘trick’’ from nature is an important componentof artificial enzyme designs.

Using this approach, artificial oxygen-activating enzymeshave been produced by the modification of native proteinscaffolds, by introducing non-native cofactors, and by theincorporation of metal cofactors into native scaffolds that donot contain metal ions.

4.1. Artificial oxygen-activating enzymes by modificationof native protein scaffolds

Fantastic progress has been made in the modification of nativeprotein scaffolds to either improve the enzyme activity, broadenthe substrate scope, or introduce new functions by engineeringnew metal-binding sites.42–45,47 There are generally two approachesfor protein design and engineering by modifications of nativeprotein scaffolds.44 One is rational design based on knowledgeof the desired chemical reaction, the original protein scaffold,and structure–function relationships from either previousexperiments or computational modeling.42,50 Although thisapproach has been successful in designing new biocatalysts, itcannot be applied in the absence of structural information onthe proteins. In this case, an alternative powerful strategy is thecombinatorial design and directed evolution of newmetalloenzymes.194–197 Directed evolution is a mimic of natural‘‘Darwinian evolution,’’ in which desired properties of proteinsare obtained by in vitro or in vivo screening of mutant librariesconstructed by random mutations, saturation mutagenesis atcertain sites or gene shuffling.194–197 For many artificial enzymedesigns, a combination of both approaches is often necessary inorder to achieve the goal. In this section, we will report relevantresults on iron- and copper-containing metalloenzymes, inwhich the oxygen activation function of the native protein hasbeen either improved or completely altered, or where newfunctionality has been engineered by modifying the proteinscaffold. Since the field is rapidly moving forward, recentadvances exploiting directed molecular evolution for the pur-poses of the design of oxygen-activating enzymes will also bedescribed.

4.1.1. Heme-proteins. One of the most successful artificialoxygen-activating enzymes produced by the modification ofnative protein scaffolds is engineered CYP, which is known to

catalyze efficient and selective C–H bond functionalizationusing O2 as an oxidant, a major challenge in both chemicaland biological catalyses.198 The use of oxygen as the oxidantalso makes these enzymes very useful for chemical transforma-tion in whole cells through fermentation.199–201 More than11 500 distinct CYPs have been identified. Despite variationsin amino acid sequences, different CYPs utilize the sameconserved structural fold to hydroxylate a wide range of sub-strates. This generalizability of CYPs, along with the potentialof their use in whole cell production of chemicals, have led tohighly focused efforts in tuning their substrate binding pocket,thus expanding their substrate scope.202,203 The overall goal isto develop CYPs capable of performing highly selective C–Hbond functionalization on any substrate of interest.

The resting state of CYP is the unreactive H2O-ligated low-spin ferric state; the binding of substrates, typically hydro-carbons, causes the transition of heme iron from the low-spinto the high-spin state and excludes water molecules from thesite. These changes result in increasing the reduction potentialof the heme iron, making it possible for the resting FeIII heme tobe reduced by its redox partner, and thus allow oxygen bindingand activation as the initial step of oxidative catalysis.199–201 As aresult of this requirement of electron transfer, almost all CYPsrequire a reductase to deliver electrons in order to function,which makes it difficult for industrial applications, because aseparate reductase has to be produced in order to couple theelectron transfer step with O2 activation. To overcome thislimitation, a soluble, catalytically self-sufficient monooxygenasefrom Bacillus megaterium called P450BM3 has been engineeredby fusing an N-terminal heme domain (BMP) to a C-terminalFAD/FMN containing reductase domain (BMR) in a singlepolypeptide chain.204 The resulting enzyme complex performshydroxylation of long chain (C12–C20) fatty acids, and theiralcohols and amides, and the epoxydation of unsaturated fattyacids with the highest catalytic activity determined for a P450monooxygenase (17 000 turnovers per min),205 due to efficientelectron transfer between the reductase and the heme.204

Because the crystal structure of P450BM3 in complex with thesubstrate is available, and since the enzyme complex expressesextremely well in E. coli, it represents an excellent platformfor biocatalysis. Therefore, huge progress has been madeby protein engineering techniques, such as site-directed muta-genesis, site-saturation mutagenesis, directed evolution or acombination of the three approaches, in enabling the creationof a wide range of P450BM3 variants with novel substrateselectivity.202–204,206–210

Directed evolution on P450BM3 has been used by Arnoldand coworkers to increase substrate promiscuity of CYPstoward short-chain alkanes, such as ethane and propane, withstrong C–H bonds.198,206,207 With the ultimate aim of engineeringan ethane hydroxylase, the authors adopted an evolution strategywhere they accumulated multiple generation of random as wellas site-saturated mutagenesis and screening.206 The authorsconcentrated on the high-resolution crystal structure of enzymebound to the palmitoglycine substrate and chose amino acidresidues that directly interact and bind either the heme or the



substrate (Fig. 10). One of the mutants selected from thisapproach, called 53-5H, was shown to catalyze 5000 turnoversof propane hydroxylation, at a rate of 370 min�1, and at least 8000turnovers of octane hydroxylation, at a rate of 660 min�1. Notably,the mutant 53-5H hydroxylated ethane to form ethanol as the soleproduct, albeit with a very slow rate of 0.4 min�1. Interestingly,53-5H contained three active site mutations A78F, A82S andA328F, all of which replace alanine with a larger side chain andpresumably reduce the volume of the active site and positionsmall alkanes above the heme during catalysis.206

Even though 53-5H attained thousands of turnovers in vitro,the utility of this catalyst remained limited because of its poorperformance to produce products in whole cells, which wasmostly due to the low efficiencies for coupling the productformation to cofactor consumption (17.4% for propane and0.01% for ethane oxidation). To overcome this limitation, theauthors used a domain-based protein-engineering strategy, inwhich the heme, flavin mononucleotide (FMN), and flavinadenine dinucleotide (FAD) domains of the P450BM3 variantwere evolved separately in the context of the holo-enzyme,and beneficial mutations were recombined in a final step.The enzyme P450PMO R2 obtained through the above approachhad 11 mutations, most of which were clustered around the FADdomain and the nearby linker of the FMN. Previous chemical and

thermal denaturation studies have shown that FMN is the mostweakly bound cofactor in P450BM3, and thus the mutationsimproved the coupling between various cofactors, and, in turn,improved the overall enzyme activity and robustness. The enzymeP450PMO R2, with improved activity and stability, not onlysupported more than 45 000 turnovers for propane hydroxylationin vitro, but also performed whole-cell biohydroxylation of propaneat room temperature and pressure with air as an oxidant.207

To exploit the potential of P450BM3 towards hydroxylationof drugs or polycyclic aromatic hydrocarbons (PAHs), Gilardiand coworkers used random mutagenesis to generate a libraryof mutants with novel activities.208–210 A double mutant, calledA2, containing the Asp251Gly/Gln307His mutations, was foundto catalyze specific hydroxylations of diclofenac, ibuprofen andtolbutamide.208 A2 was able to bind and turn over tolbutamidewith rate constants higher than those measured for ibuprofenand diclofenac (kcat = 0.94 min�1, kcat = 0.10 min�1, andkcat = 0.048 min�1, for tolbutamide, ibuprofen and diclofenac,respectively). Even though the catalytic activity for A2 was lowwhen compared to the values calculated for the known sub-strate lauric acid (kcat = 1810 min�1 for WT P450BM3, and kcat =697 min�1 for A2, respectively), new binding and catalyticabilities have been introduced in the enzyme by mutating onlytwo amino acids in positions not directly involved in substratebinding or turnover.

In subsequent studies, the authors used directed evolutionof P450BM3 for the recognition and oxidation of chrysene andpyrene.209 Three different mutants, named M3, P2 and K4,showed higher affinity and coupling efficiency for both sub-strates with faster rates of product formation compared withthe wild-type enzyme. DNA sequencing of the mutants revealedthe presence of 6 mutations in M3 (Asp251Gly, His266Ala,Glu267Arg, Thr269Asn, Ser270Glu, and Gln307His), 4 muta-tions in P2 (Asp208Arg, Ile209Tyr, Asp251Gly, and Gln307His),and only one single mutation, Val317Cys for K4. Measurementsof the performance in terms of the reaction rate and relativecoupling efficiency of the mutants and WT were carried out inreference to the production of 6-hydroxychrysene and 1-hydroxy-pyrene, respectively. Comparison between the data obtainedfrom the mutants M3, P2 and K4 and chrysene and pyreneshowed that both the rate of product formation and relativecoupling efficiencies (calculated from the ratio betweenhydroxyl-product formed and NADPH consumed) were higherfor pyrene than chrysene. These results are impressive demon-strations of how directed evolution may provide enzymes withnew biocatalytic capabilities. This approach represents a powerfultool for producing protein variants with mutations in randompositions, even far from the active site, otherwise unpredicted toaffect the enzyme functionality by using a rational approach.

Site-directed mutagenesis is an excellent strategy to increasethe substrate scope of CYPs; however, it often results in a loweryield and stability than those of the native enzyme. As analternate strategy, Reetz211 and Watanabe212–214 groups haveused small decoy molecules into ‘‘fooling’’ CYPs to catalyzeC–H bond functionalization on non-native substrates. The crystalstructure of palmitoleic acid bound P450BM3 shows that the

Fig. 10 (a) Substrate channel and (b) active site residues targeted forsaturation mutagenesis mapped on the palmitate-bound structure of theP450BM3 heme domain (PDB ID: 1fag). Heme (white) and fatty acid(orange) are shown in space-filling mode. (c) H-bonding interactionsbinding the palmitoleic acid substrate in P450BM3. Adapted fromref. 207, Copyright 2007 Wiley-VCH.



substrate is fixed by two major interactions: (1) hydrophobicinteraction of the alkyl chain with amino acids at the substratebinding site and (2) interaction of the substrate carboxylategroup with Tyr51 and Arg47 (Fig. 10c). Both of these inter-actions were required for the proper placement of the substratein the active site of enzyme such that the enzyme is triggered‘on’, reduced and then binds O2 at the heme iron to performthe catalytic C–H bond oxidation. Building on this observation,Watanabe and coworkers designed perfluorinated versions oflong chain fatty acid substrates (called PFCs) as they would‘‘trick’’ CYPs into believing that the substrate was recognizedand hence remain switched on for catalytic hydroxylation ofnon-native substrates (Fig. 11). The PFCs were expected toinitiate the activation of molecular O2 in the same manner asthe long-alkyl-chain fatty acids and induce the generationof Compound I (the key oxidizing species in CYPs) to oxidizenon-native substrates like propane, while PFCs will never beoxidized due to their stronger C–F bonds (B116 kcal mol�1).The first generation PFCs, thus designed, contained 9–13 carbonatoms giving enough space for the binding of propane at thecatalytic heme center. As was expected, the product formationrates were highly dependent on the alkyl-chain length of PFCs,with PFC10 displaying the highest rate of product formation(67 min�1 for propane).213 Building upon previous substrateoccupancy studies on CYPs, the authors designed decoy mole-cules with increased H-bonding interactions at the substratebinding site. These second generation PFCs not only showed atleast 100-fold improved binding affinity to P450BM3 but alsoincreased the turnover rates to 256 min�1 for propane and45 min�1 for ethane.214 Most importantly, there were no over-oxidation products for these transformations, which encouragedthe authors to investigate the hydroxylation of aromatic com-pounds using a series of PFCs (PFC8–PFC12). PFC9, in particular,afforded the largest turnover rate of 120 min�1 indicating that theactive site provided by PFC9 was suitable for the accommodationof benzene. Interestingly, the o-position of monosubstitutedbenzenes was selectively hydroxylated, regardless of the substi-tuents, suggesting that the structural recognition is responsiblefor the selectivity of CYP reaction.212 At the same time, PFCsof different chain lengths were screened to obtain a suitableactivator for selective hydroxylation of propane and butane isomers.Remarkably, the PFC of formula CF3(CF2)7COOH could hydro-xylate methane to methanol, the ‘‘holy grail’’ of C–H bondfunctionalization through dioxygen activation, displaying morethan 2000 turnovers.211 These results on CYPs suggest that theuse of decoy molecules is an excellent technique to alleviateenzymes from their dependence on substrate cofeeds andexpand their substrate scope.

Moreover, decoy molecules can eventually be combined withtraditional mutagenesis on the target enzyme to increase itscatalytic efficiency. The CYPs are extremely versatile in that theynot only perform selective C–H bond hydroxylation but alsoother transformations including epoxidation, oxidative defor-mylation, dehydrogenation, rearrangements, Baeyer–Villigeroxygenation, and oxidative decarboxylation.215 Recently, a novelclass of nitrating CYP TxtE have also been discovered that

catalyze the direct and regioselective aromatic nitration usingoxygen and nitric oxide as substrates.216 This generalizability inthe CYP reactivity has encouraged researchers to expand itsreactivity (through directed evolution and replacement of theaxial cysteine residue) to include different transformations likehalogenation, cyclopropanation, N–H insertion, C–H aminationand sulfimination.217,218 However, these reactions are beyondthe scope of this review and readers are referred to a recentreview focusing on non-natural reactions by CYPs.202,219

The example of CYPs discussed above shows how a proteincan be engineered to perform chemo- and site-selective catalysison different substrates. Engineering and redesigning a protein tomodel structural units of another protein also help to under-stand the structure–function relationship in the latter in greaterdetail and result in different applications, such as alternativeenergy productions. A notable example of that is the redesign ofmyoglobin to mimic the catalytic center of cytochrome c oxidase(CcO). CcO is a trans-membrane protein, which catalyzes thefour-electron reduction of oxygen to water during aerobicrespiration. The catalytic center of CcOs consists of a heme–coppercenter, where the copper is coordinated to three histidines with one

Fig. 11 Schematic of the reaction mechanisms of (a) the natural reactionsystem and (b) the decoy molecule system of P450BM3. (a) Subterminalcarbons of fatty acids are hydroxylated in the natural reaction. (b) By simpleaddition of a decoy molecule such as PFC9, the hydroxylation reaction of asmall alkane (for example, propane) is catalyzed by P450BM3 because ofsubstrate misrecognition of P450BM3. Reprinted with permission fromref. 212, Copyright 2015 Wiley.



of the histidine crosslinked to a tyrosine residue (Fig. 12).220,221

Despite decades of investigation of oxygen reduction in CcOs,structural features responsible for the efficient reaction are notwell understood. This gap in understanding is primarily due to thefeatures of the membranous CcO protein as well as to the presenceof multiple metal cofactors (for example a CuA and a heme centerthat deliver electrons to the heme–copper active site where O2 isreduced) in the enzyme complex, which makes its spectroscopicinvestigation very challenging. Furthermore, CcO is also a modelcatalyst for oxygen reduction reaction (ORR) in fuel cells, as it usesearth abundant metal ions and has a much lower overpotentialthan the current state-of-the-art platimum-based ORR catalysts.However, the large size and low stability of the CcO make difficultits application as an ORR catalyst. In order to overcome thesechallenges, a number of small molecule-based synthetic modelsof CcO have been developed and these systems have providedtremendous knowledge about the structure and functions ofCcOs,222,223 even though most of the models have low activityand turnover numbers.

To complement the synthetic modeling approach, Lu andcoworkers have developed an alternative method to study theoxygen reduction catalysis in CcOs by designing a heme–coppercenter in a small, easy-to-purify heme-containing soluble protein,myoglobin, that is free of other metal cofactors (Fig. 12). The firstgeneration models focused on designing a copper center distal tothe histidine coordinated high-spin heme. To engineer a copperbinding center, two histidines (L29H, F43H) were introduced inthe distal pocket of myoglobin that, along with the third histidinealready present (H64), could potentially bind copper similarto CcOs; this myoglobin mutant was named CuBMb.224–226 Thecopper binding in the distal pocket of the heme centerwas confirmed by several spectroscopic and crystallographicmethods.224 The introduction of the copper binding center,while increased the product selectivity of the oxygen reduction(the ratio of water produced with respect to reactive oxygenspecies such as superoxide and peroxide) with respect toWTMb, also lowered the total rate of oxygen reduction. Toimprove the activity and product selectivity of the CcO model,tyrosines were engineered close to the heme–copper catalytic

center, thus affording F33Y-CuBMb and G65Y-CuBMb, whichnot only improved the activity and product selectivity (B80%water production), but also performed more than a thousandturnovers (Fig. 13).227 In fact, by increasing the electron transferrates to the heme cofactor using the native electron transferpartner of Mb (cyt. b5), the G65Y-CuBMb variant was shown toreduce oxygen with rates (52 s�1) comparable to a native CcO(50 s�1).228 Moreover, when the designed protein was immobi-lized onto electrodes, the electrocatalytic reduction of oxygenusing G65Y-CuBMb not only resulted in very selective oxygenreduction (B96% water) but also in exhibited rates (5000 s�1)exceeding the fastest CcO (bovine CcO with a rate of 500 s�1).229

These results demonstrated that it is possible to redesign themetal-binding sites in native scaffolds that display new func-tions (e.g. from reversible O2 binding in WTMb to catalytic O2

reduction to H2O) that can meet or even exceed the catalyticefficiency of native enzymes.

The rationally designed functional mimics of CcO thusprovided an efficient method to probe and understand the roleof various structural units, such as the crosslinked tyrosineconserved in all CcOs. To elucidate the role of tyrosine in oxygenreduction, unnatural tyrosine variants, such as monochloro-,dichloro-, fluoro- and methoxy-tyrosines, were incorporatedgenetically at the 33 position of the functional CcO mimic, F33Y-CuBMb (Fig. 13).230 The oxygen reduction activity measurements

Fig. 12 (a) The heme–copper catalytic center of cbb3 oxidase with theCuB coordinated to three histidines with one of the histidines crosslinkedto tyrosine (PDB ID: 3mk7). (b) The designed heme–copper center in theCcO mimic in myoglobin (F33Y-CuBMb) containing the CuB coordinatedto three histidines. A Tyr33 has also been incorporated into the active siteto mimic the conserved Tyr in CcOs (PDB ID: 4fwy).

Fig. 13 (a) Correlation of the oxidase activity of Phe33Tyr–CuBMb,Phe33ClY–CuBMb, Phe33F2Y–CuBMb, and Phe33F3Y–CuBMb vs. pKa ofphenols on the Tyr and its analogs. (b) Correlation of water produced inoxygen reduction reaction performed by these proteins vs. the pKa ofphenols on the Tyr and its analogs. (c) Correlation of oxidase activity bythese proteins vs. peak potential at pH 13 (Ep) of the corresponding Tyr andTyr analogs. The structures of various Tyr analogues used in this study aredisplayed in the left. Adapted with permission from ref. 228, Copyright2015 American Chemical Society.



showed a systematic increase in the oxidase activity of the CcOmimics with an increase in Tyr redox potential and a decrease inTyr pKa, strongly suggesting the role of tyrosine as an H radicaldonor to oxygen for its reduction. To prove the role of Tyr andobserve oxygen reduction intermediates in the CcO mimic, thereduced F33Y-CuBMb was mixed with oxygen and the reactionmixture, freeze-quenched at a short timescale (B20 ms), andprobed via continuous wave-EPR. The experiments revealed theformation of a Tyr radical under reaction conditions, which forthe first time, established the role of Tyr as an H radical donor tooxygen in CcOs.231

Another unique structural feature of CcOs is the presence ofdifferent heme types at its catalytic center. While, A- and B-typeoxidases contain hemes a and o, the C-type oxidases containheme b (Fig. 14).232 An efficient method to understand the roleof different heme types in the function and the stability of CcOswould be to replace different heme types and studying theirimpact on enzymatic activity. However, the presence of differentheme cofactors (e.g. the heme that delivers electrons to theheme–copper center) and the membranous nature of CcO makesuch a study very difficult, if not impossible. The Lu groupaddressed this issue, by incorporating mimics of heme a andheme o into the designed CcO mimic.233 The incorporation of aheme variant into the hydroxyl group attached to the porphyrincore (similar to heme o) increased the stability of CuBMb bydecreasing the rate of heme degradation by approximately19-fold.233 Furthermore, the incorporation of heme a mimics(with one or two formyl groups conjugated to the porphyrin core)increased the heme redox potential and in turn the overall rate ofoxygen reduction by more than 6-fold (Fig. 14g).234 Overall, thisstudy reveals that the CcO uses different heme types such asheme a and heme o to control the heme redox potential, oxygenreduction activity and stability of the enzyme.

Apart from designing the Cu binding site in Mb, Lu andcoworkers have also designed a Mn binding site in cytochromec peroxidase (CcP), to mimic the function of manganese peroxidase(MnP).235 The MnP from the white rot fungus P. chrysosporiumplays a vital role in lignin degradation. MnP binds two CaII, oneMnII and a heme FeIII that oxidize MnII to MnIII via hydrogenperoxide activation. The enzyme-generated MnIII is subse-quently used to oxidize organic substrates. To test the currentunderstanding of the structure and the function of MnP and tofind an alternative catalyst for oxidative delignification, theauthors designed and engineered a MnII binding site similar toMnP in easy-to-work-with peroxidase, CcP, that can be readilyexpressed in E. coli in high yields.236 The CcP mutant (MnCcP),thus created, based on structural comparisons and computermodeling, binds MnII in a manner similar to the native enzymeand shows that the incorporation of the MnII binding sitefacilitates MnII oxidation. Further mutations of the residuesin the heme distal pocket (W191 and W51) in MnCcP to thecorresponding Phe in MnP conferred even higher MnP activityof the protein model. Interestingly, the two mutations do notcontribute equally to the activity increase. A much largerincrease arises from the W51F mutation because W51 stabilizesthe heme compound II (oxo-ferryl heme species). Since, the

reaction of compound II with MnII is rate-limiting, a morereactive compound II increases MnP activity.237

Despite the above progress made in redesigning CcP intoMnP, most of the designed enzymes still suffer from relativelylow activity and moderate to low binding affinities for the metalthan those in native MnP. It was hypothesized that one reasonfor such low activity and metal-binding affinity is our lack ofunderstanding about the subtle secondary coordination sphereinteractions around the active site that are responsible forfine-tuning the activity.34 While these interactions, such ashydrogen-bonding, salt bridges to the metal ion ligands, hydro-phobicity of the site, and overall rigidity or flexibility of the site,have been recognized to be important, few studies have demon-strated engineering these interactions to improve enzymeactivity and metal-binding affinity.110,162,165,170 To address theabove issue, specific secondary sphere residues around theMnII-binding site of the engineered MnCcP (MnCcP1) wererationally mutated. This resulted in improved binding affinityto MnII and/or catalytic efficiency toward MnII oxidation anddegradation of lignin (Fig. 15).238 First, removing a hydrogenbond to Glu45 through Tyr36Phe mutation enhanced MnII-binding affinity, as evidenced by a decrease of KM of MnII

Fig. 14 (a) Protein scaffold of F33Y-CuBMb. (b) Heme b cofactor presentin F33Y-CuBMb. (c) Heme a present in the catalytic site of bovine CcO.(d) Diacetyl heme. (e) Monoformyl heme. (f) Diformyl heme incorporatedinto F33Y-CuBMb apo-protein. (g) Variation of oxygen reduction activitywith heme E1 for F33YCuBMb variants. The dotted blue line indicates E1of WTMb and bovine CcO. Reprinted with permission from ref. 234,Copyright 2014 American Chemical Society.



oxidation 2.8-fold. Second, introducing a salt bridge throughLys179Arg improved Glu35 and Glu181 coordination of MnII,decreasing KM 2.6-fold. Third, eliminating a steric clash thatprevented Glu37 from orienting towards MnII resulted in an8.6-fold increase in kcat/KM arising primarily from a 3.6-folddecrease in KM. This effort has resulted in one variant (Ile40Gly)displaying a KM value that is comparable to that of native MnPs.(0.28 mM vs. 0.2 mM for Pleurotus eryngii MnP PS3). This workfurther demonstrated that while the effects of Tyr36Phe andLys179Arg mutations are additive, other combinations of muta-tions were antagonistic. Using both alkali treated lignin and alignin model compound, the variants were also shown to befunctional mimics of MnPs. This study not only provides abetter functional model of MnP, but more importantly it alsoconfirmed the importance of the secondary coordination inter-actions to enhance metal-binding affinities and overall activity.

4.1.2. Copper proteins. Copper proteins fulfill importantroles in nature, including catalytic oxygen activation. Thereforeit is not surprising that they have been the subject of manyprotein engineering studies, wherein the native proteins arealtered in an attempt to improve their functionality. Severalapproaches have been employed depending on the proteinfamily under study. For example, in the case of MCOs, directedevolution has been preferred over rational design approaches,due to the lack of detailed structural information on theseenzymes.239

Tyrosinases are copper-containing enzymes widely distributedin Nature, which utilize dioxygen to catalyze two successiveenzymatic reactions: (i) the ortho-hydroxylation of monophenolsto ortho-diphenols (monophenolase activity) and (ii) the oxidationof ortho-diphenols to ortho-quinones (diphenolase activity).240

These enzymes have a strong preference for phenolic anddiphenolic substrates and are somewhat limited in their reactionscope, always producing an activated quinone as a product.Despite this fact, they can be potentially utilized in several

biotechnological applications, including the production ofL-DOPA, phenol and novel mixed melanins, biosensors ofphenols, and removal of dye and other compounds.241 Therefore,many efforts have been devoted to the applications of directedevolution methods to engineer improved tyrosinases. Fishmanand coworkers used directed evolution on tyrosinase from Bacillusmegaterium (TyrBm) in an attempt to improve its monophenolase/diphenolase activity ratio.242 Through this approach, they dis-covered one variant, R209H, displaying a 1.7-fold improvementin monophenolase activity and a 1.5 decrease in diphenolaseactivity, with an overall 2.6-fold improvement in the mono-phenolase/diphenolase activity ratio. The authors hypothesizedthat the imidazole group of the newly introduced histidineresidue obstructs the entrance to the active site, thus inter-fering with the binding of L-DOPA to one of the two copper ions.Fig. 16 illustrates the active site structures for both wild-typeTyrBm (Fig. 16a) and variant R209H (Fig. 16b).243 Comparisonof wild-type structures with the structure of the site-specificvariant R209H, which possesses a higher monophenolase/diphenolase activity ratio, lent further support to the hypothe-sized mechanism by which monophenolic substrates dockmainly to CuA. The Arg209 residue is positioned in proximityto the entrance of the active site, on helix a6 adjacent to His208,which coordinates CuB. In subunit 1 of TyrBm, two conformationsof this residue were observed, indicating its flexibility (Fig. 16a). Inthe R209H TyrBm mutant structure, His209 can be seen to shieldCuB and to obstruct the entrance to the active site (Fig. 16b). Thisstructural evidence further supports the hydroxylation mechanismin which a monophenol substrate docks to CuA. This work clearlyhighlights the potential of the directed evolution approach tomodulate the function of native proteins, and it is of greatrelevance since the hypothetical mechanism has been thenconfirmed by structural data.243

Among the MCO families, laccases represent the most attrac-tive enzymes due to their possible application in bioremediation,organic synthesis, biosensors and other industrial applications.They show broad substrate specificity and relative autonomy,since they use molecular oxygen from air as an electron acceptorand they only produce water as the by-product.17 They couple thefour single-electron oxidations of the reducing substrate to the

Fig. 15 Secondary shell residues rationally mutated in MnCcP.1 and theireffect on activity and metal affinity. Reprinted with permission fromref. 238, Copyright 2016 American Chemical Society.

Fig. 16 The active sites of wild-type TyrBm and variant R209H. (a) Theactive site of wild-type TyrBm (TyrBm1) is presented in blue, and the twoconformations of Arg209 identified in the electron density maps arepresented in yellow. (b) The active site of variant R209H (structure ofTyrBm1_mut) and residue His209 is shown in light blue and yellow,respectively. Reprinted with permission from ref. 243, Copyright 2011Elsevier.



four electron reductive cleavage of the dioxygen bond, using fourCu atoms distributed over three sites.15,16 Typical metal contentof laccases includes one type 1 copper (T1Cu), and one type 2copper (T2Cu) and two type 3 copper ions (T3Cu), with T2Cu andT3Cu arranged in a trinuclear cluster (TNC).244 The range offunctions exhibited by laccases is broadly divided into threecategories: (1) cross-linking of monomers, (2) degradation ofpolymers, and (3) ring cleavage of aromatic compounds. In orderto highlight the power of directed evolution approaches in thedevelopment of copper-based oxygen-activating enzymes, severalselected examples of engineered fungal laccases are described inthe following.245,246 However, for a more detailed description oflaccases, their properties and their applications, the reader canrefer to previous excellent reviews on this topic.244,247,248

The first successful example of directed evolution to modifya fungal laccase was reported by Arnold and coworkers.249 After10 rounds of selection, they obtained a laccase variant with14 mutations accounting for a 170-fold improvement in totalactivity, an 8-fold increase in the expression levels and 22-foldin the kcat for ABTS. Among laccases, those possessing highredox potentials at the T1 copper site (ranging from +430 mVin bacterial and plant laccases, to +790 mV in some fungallaccases) are classified as high redox potential laccases(HRPLs). They are typically secreted by ligninolytic basidio-mycetes (white-rot fungi), and are excellent candidates forindustrial applications, since their high redox-potential valuesallows them to oxidize a wide variety of substrates.245,250

Thermostable laccases with a high redox potential have beenengineered by Mate and coworkers,251 through a strategy thatcombines directed evolution with rational approaches. Thestarting point was the basidiomycete PM1 HRPL functionallyexpressed in S. cerevisiae, which exhibits a remarkable stabilityand activity, including thermal activation. After eight rounds ofevolution, the total laccase activity was enhanced 34 000-fold,obtaining the OB-1 mutant as the last variant of the evolutionprocess, displaying a highly active and stable enzyme in termsof temperature, pH range, and organic cosolvents.251 Similarly,Camarero and coworkers described a directed-evolution platformfor the development of Pycnoporus cinnabarinus laccase (PcL) withdifferent properties.252 After six rounds of evolution coupled tohigh-throughput (HTP) assays based on the oxidation of naturaland synthetic mediators, the total laccase activity was improved8000 times. The final mutant of this study (the 3PO variant)accumulated a total of 15 mutations in the fusion gene. Fivemutations were responsible for a 40-fold enhancement insecretion by S. cerevisiae (B2 mg L�1), while the ten beneficialmutations in the mature protein led to a 13.7-fold increase inthe kcat for ABTS.252 Semi-rational mutagenesis of the laccasePOXA1b from Pleurotus ostreatus was performed through acombination of directed evolution with elements of rationalenzyme modification, by Sannia and coworkers.253–255 The finalevolved mutant, containing five non-synonymous mutations,increased 3.5-fold in its activity towards ABTS and 2-foldtowards 2,6-dimethoxyphenol (DMP), with higher affinity towardsDMP. A significant change in the laccase selectivity towardsthe oxidation of phenolic substrates has been obtained by

Wang and coworkers, who prepared several mutants ofKlebsiella sp. 601 MCO.256 They found that the a-helix (fromLeu351 to Gly378) covering the substrate binding pocketstrongly influences substrate selectivity, probably by modulatingthe electron transfer between the substrate and the T1 coppersite. Seven mutations were introduced in this helical fragment,affording the a351-380M mutant. These mutations seem todisrupt the helix secondary structure, thus enhancing specificitytoward the phenolic substrate DMP. This finding makes themutant very attractive for applications in biotechnology andgreen chemistry.

Azurin from Pseudomonas aeruginosa is a type 1 copperprotein involved in biological electron transfer. It has been asubject of extensive protein design and engineering to under-stand structural features responsible for efficient electrontransfer.257,258 Recently, it has been used by the Lu group tounderstand post-translational modification of cysteinyl thiolateto sulfonate, which plays important roles in biology, such asredox signalling, enzyme and gene regulation, by designing anazurin cavity mutant (M121G-Az) that makes the copper centeraccessible to small molecules like hydrogen peroxide andoxygen.259 The reaction of hydrogen peroxide with CuI-M121G-Azresulted in the first copper-sulfenate characterized in a proteinenvironment, as supported by resonance Raman spectroscopy,electrospray mass spectrometry using isotopically enrichedhydrogen peroxide, and density functional theory calculationscorrelated with the experimental data. Furthermore, structuraland computational studies suggested that in the secondarycoordination sphere non-covalent interactions played animportant role in stabilizing this highly reactive species, whichcan further react with oxygen to form a sulfinate and then asulfonate species, as demonstrated by mass spectrometry.Engineering the electron transfer protein azurin into an activecopper enzyme that forms a copper-sulfenate center and demon-strating the importance of non-covalent secondary sphere inter-actions in stabilizing it are significant in understanding themetal-sulfenate species in biological systems.

4.2. Artificial oxygen-activation enzymes by cofactorreplacement

To expand the repertoire of metalloenzyme functions and todesign new artificial molecules, the strategy of replacing thenatural cofactor housed in a native protein has been reportedsince the 1980s.260 Towards this aim, small molecule-basedchemical catalysts show some interesting properties.261 Theycan be produced in high quantity and at low cost throughchemical synthesis, and possess high stability and resistance tosevere conditions of temperatures and pressures. However,compared to natural biocatalysts, chemical catalysts often showlow selectivity and turnover numbers, due to the lack of anappropriate molecular environment, which controls both thefirst and secondary coordination spheres. This disadvantagecan be overcome by inserting a chemical catalyst within aprotein environment.261,262

Heme-proteins are particularly well suited for cofactorreplacement, since the heme pocket represents a suitable protein



cavity for inserting an unnatural cofactor. An intricate set ofdifferent interactions allows heme to be held in the proteinmatrix. These interactions create a molecular architecture aroundthe cofactor, which contribute to a fine tuning of the hemeproperties. The number, types and donor properties of axialligands directly contribute to heme properties.34 Hydrophobic,ion-pairing and hydrogen-bonding interactions dominate thesecondary coordination sphere and control several features ofthe heme environment, such as the local dielectric constant,heme exposure, and the ligand orientation. Furthermore, theprotein regulates the accessibility of solvent and substrates intothe active site, thus allowing the selective discrimination betweendifferent ligands and/or substrates. Replacing the heme with anunnatural cofactor can be facilitated by the enormous amount ofstructural data available on natural and mutated heme-proteins:a careful inspection of the pocket structure at a molecular levelcan suggest that the cofactor can be inserted and mutations canbe performed for housing that cofactor.

Through this approach, Hayashi and coworkers reportedthe engineering of new functions inside myoglobin (Mb)(Fig. 17a).263,264 Myoglobin is a dioxygen binding protein andlacks a recognition site of substrates different from dioxygen inthe heme proximity. Thus, the first aim was to provide myoglobinwith a substrate binding pocket. Aryl substituents were intro-duced on the propionate groups of protoheme IX, thus giving theunnatural cofactor 2, shown in Fig. 17b. Mb reconstitution withthis unnatural cofactor afforded a peroxidase-like catalyst, whichwas able to activate hydrogen peroxide and catalyze guaiacoloxidation with an efficiency 13-fold higher than native myoglobin.Next, in order to increase the catalytic properties of this molecule,some key amino acid mutations were introduced in the myoglobinstructure.265 In particular, mutation of the distal His64 to Asp,

aimed at expanding the substrate binding pocket, into myoglobinresulted in a 430-fold enhancement of the catalytic efficiency(kcat/KM) toward guaiacol oxidation with respect to WT myoglobin.In an effort to increase the kcat value, modified hemes substitutedat only one of the two propionates (cofactors 3a and 3b, Fig. 17b)were prepared. Reconstitution of MbH64D with these cofactorsresulted in an artificial enzyme with an increased kcat value withrespect to MbH64D-1 (MbH64D-3 kcat = 24 s�1; MbH64D-1kcat = 1.2 s�1) and with a catalytic efficiency comparable to thatof native HRP (MbH64D-3 kcat/KM = 85 000 M�1 s�1; HRP kcat/KM = 72 000 M�1 s�1).118

These results demonstrated that the replacement of thenative heme with an artificially synthesized heme is an attractivestrategy to regulate the myoglobin function. In subsequentstudies, the authors tried to increase the peroxidase andperoxygenase activity of myoglobin by modifying the tetra-pyrrolic framework of the prosthetic group, using porphyrinoids.Towards this aim, porphycene (Pc), a constitutional porphyrinisomer coordinated by FeIII, was used (Fig. 17c).266 As revealedfrom the analysis of the crystal structure, the FeIIIPc cofactor wasbound to the protein scaffold situated between the E and Fhelices, and with the His93 of the F helix as a fifth ligand.Spectroscopic analysis of the FeIIIPc reconstituted myoglobinrevealed that the unnatural cofactor is more tightly coordinatedto His93 with respect to WT myoglobin. The authors suggestedthat the strong coordination of the Fe–His93 bond in thereconstituted FeIIIPc–myoglobin contributes to the enhancementof the catalytic activity toward the oxidation of substrates suchas guaiacol, thioanisole, and styrene, with respect to nativemyoglobin. At pH 7.0 and 20 1C, the initial rate of the guaiacoloxidation was found to be 11-fold faster than that observed forthe native myoglobin.

Fig. 17 Myoglobin reconstitution with an artificial cofactor for inserting new functions: (a) General strategy for myoglobin and its mutant reconstitution.Adapted from ref. 265, Copyright 2004 American Chemical Society. (b) Molecular structures of the modified hemes. (c) Molecular structure ofporphycene (Pc).



Because manganese porphyrins and porphyrinoids such asmanganese corrole and corrolazine have been studied as catalystsfor C–H bond activation, more recently the authors usedmyoglobin reconstituted with the MnIIIPc cofactor to obtainan hydroxylation catalyst.267 As observed from the solution ofthe crystal structure of the reconstituted protein, obtained at2.2 Å resolution (Fig. 18), and as already observed for the ironcomplex, MnIIIPc is coordinated by His93, thus being accom-modated into the natural heme-binding site. Upon addition ofH2O2 at pH 8.5, rMb(MnPc) promotes hydroxylation of ethyl-benzene with a total turnover number (TON) of 13 and an initialturnover frequency (TOF) of 33 h�1, yielding 1-phenylethanol asa single product.267 The finding that myoglobin, only whenreconstituted with MnPc, is able to catalyze hydroxylationreactions is clearly related to the presence of MnIII. In addition,the direct involvement of the metal ion-center into the hydroxylationreaction mechanism was demonstrated by using O18 labeledhydrogen peroxide. The results of this work clearly demonstratethat by choosing the correct unnatural cofactor, artificialmetalloenzymes with unprecedent functional properties canbe designed.

The heme pocket of heme-proteins can also accommodatenon-tetrapyrrolic cofactors. Ueno and Watanabe first reported adetailed screening of the insertion of Schiff base metal complexesinto the protein environment by non-covalent conjugation.268

The interest in reconstituting myoglobin with Schiff base metalcomplexes derived from the finding that these complexes,containing MnIII and CrIII, have been reported as oxidationcatalysts in organic solvents.269,270 In addition, their molecularsize and coordination geometry are similar to heme, and it iseasy to modify the ligand size and hydrophobicity. The authorsreported the incorporation of several Fe, Mn and Cr salen andsalophen complexes (Fig. 19) into myoglobin and somemutants, to analyze the stereoselective oxidation of thioanisole.Detailed screening of metal-binding ability and stability,together with resolution of the crystal structures of some ofthe reconstituted proteins, allowed for the redesign of metalcomplex/protein composites with improved catalytic activityand stereoselectivity.271–274,288 From these studies, they concludedthat A71G mutation seemed to be necessary to make myoglobinsuitable for accommodating the Schiff base complexes withoutsteric bumps.

The crystal structure of Fe�2�salophen apo-A71GMb con-firmed that the iron complex Fe�2 is fixed in the heme cavity

of apo-Mb (Fig. 20a), and axially coordinated by His93.273

Specific interactions with Phe43, Leu89, His97 and Ile99influence the orientation of the Fe�2 complex in the cavity ofapo-A71GMb. The side chain of Ile107 is located between the3- and 30-methyl groups of Fe�2. Inspection of the crystalstructure of Mn�2�apo-A71GMb revealed a narrow channelbetween His64 and the phenylenediamine unit of 2. Replacementof His64 with Asp (H64D mutation) was expected to increase theaccess of substrates and oxidants to the vacant distal site abovethe metal ion-center. In addition, to further enlarge the active siteaccess, the phenylendiammine unit was replaced by ethylen-diammine affording the salen ligand series (Fig. 19b). The ratewith Mn�4�apo-H64D/A71GMb was found to be 3-fold higher thanthat with Mn�2�apo-H64D/A71GMb, thus confirming that theenlargement of the substrate cavity increases the accessibility ofthe substrate.233 Substitutions at the 3,30-positions strongly affectthe enantioselectivity of the sulfoxidation reaction. Interestingly,while Mn�4�apo-H64D/A71GMb showed 32% ee (R) selectivity,introduction of bulkier groups at the 3,30-positions inducesrelative S-selectivity to end up 13% (S) for 6. All the resultsdemonstrate that the enantioselectivity and catalytic activity ofunnatural metal cofactors in protein cavities can be regulatedby rational design of the cofactors.

An alternative approach for the site-specific attachment ofa non-natural cofactor into a protein scaffold is by covalently

Fig. 18 Cofactor replacement in myoglobin as adopted in the work byHayashi et al.267 Crystal structures of WT-myoglobin (left PDP ID: 1myo)and MnIIIPc substituted myoglobin (PDP ID: 3wi8).

Fig. 19 Schiff base complexes inserted into myoglobin: salophen ligandseries (a) and salen ligand series (b) screened for the activity.

Fig. 20 (a) Crystal structure of apo-A71GMb reconstituted with the Fe 2salophen ligand. Reprinted with permission from ref. 273, Copyright (2004)American Chemical Society. (b) (top) Salen ligand modified by methyl thio-sulfonate linking arms. (bottom) Computer model of the Mb cavity with MnSalen(green) positioned to overlap the space occupied by the native heme cofactor.The residues selected as possible anchor positions are visible. Left anchors T39and Y103 are purple, and right anchors L72 and S108 are orange. Reprinted withpermission from ref. 278, Copyright 2011 American Chemical Society.



linking the two. Towards this aim, manganese salen withmethyl thiosulfonate linking arms (Fig. 20b) was covalentlyattached to Cys residues inside the Mb cavity by the Lu group,who demonstrated that the incorporation of MnSalen into aprotein scaffold enhances the chemoselectivity in the sulfoxi-dation of thioanisole275 and that both the polarity andhydrogen-bonding of the protein scaffold play an importantrole in tuning the chemoselectivity.276 Furthermore, a comparisonof the effect of different pH values on sulfoxidation and ABTSoxidation indicates that, while the intermediate produced underlow pH conditions could only perform sulfoxidation, the inter-mediate at high pH could oxidize both sulfoxides and ABTS. Sucha fine-control of reactivity through hydrogen-bonding interactionsby the distal ligand to bind, orient and activate H2O2 is veryimportant for designing artificial enzymes with dramatic differentand tunable reactivities of catalysts without protein scaffolds.277

Importantly, the effect of different anchoring positions onreactivity and selectivity was investigated.278 This metalloenzymedesign started by modeling the salen ligand into the myoglobinstructure and searching for the best position for covalent anchoringthe cofactor in a location similar to the heme in the proteinbinding pocket. The alignment of the metals and donor atoms ofthe heme and the model salen complex allowed the positioningof Mn–salen in the heme-binding site. The calculation suggestedT39, L72, Y103 and S108 as appropriate anchoring groups to linkthe salen ligand in a position similar to the native cofactor (seeFig. 20b). Four myoglobin mutants Y103C/S108C, T39C/S108C,Y103C/L72C, and T39C/L72C carrying two Cys residues necessaryfor the covalent linkage of the salen ligand arms to the proteinwere expressed and purified. Next, the effects of the differentanchoring positions on the reactivity of the reconstituted proteinstoward thioanisole sulfoxidation were analyzed. In order to distin-guish between the impact of the different anchoring positions,anchor residues were clustered as belonging to the right side (L72or S108) or left side (T39 or Y103) with respect to the salen ligand(see Fig. 20b). Interestingly, the anchor position of the salenligand strongly affects both the reaction rate and selectivity, andthe effects are additive in nature and independent. For example,changing the anchor positions, from 1�Mb(Y103C/S108C) to1�Mb(T39C/L72C), results in total rate enhancements of 7-foldand an 80% increase in selectivity for the S enantiomer. Togain more insight into the molecular basis for the observedselectivity, the reactivity of the reconstituted Mbs was assayedtoward a series of substituted aryl methyl sulfides, withdifferent substituents. The right anchor site was observed tohave a greater influence than the left anchor site on thereactivity and selectivity in sulfoxidation, and 1�Mb(T39C/L72C) showed the highest reactivity (TON up to 2.32 min�1)and selectivity (ee% up to 83%) among the different anchoringpositions examined. Molecular dynamic simulations revealeddifferences in the substrate access path for the L72C anchorposition; steric effects inside the protein cavity force thesubstrates to enter in a different way limiting the conformationalmobility, thus enhancing stereoselectivity. Overall, this work high-lights that attachment strategies may have considerable influenceon the activities of non-native cofactors.

Simple replacement of the metal ion has also been shown tobe effective in providing new enzymes with oxygen-activatingactivity. Several examples deal with the replacement of theZnII ion with redox active ions, such as MnIII and CuII.279–281

Substitution of manganese for zinc in carbonic anhydrase (CA)converted this hydrolase to an enantioselective peroxidase.280

Manganese-substituted carbonic anhydrase (CA[Mn]) showsperoxidase activity with a bicarbonate-dependent mechanismtowards the oxidation of o-dianisidine with kcat/KM = 1.4 �106 M�1 s�1. In addition, CA[Mn] also catalyzed the moderatelyenantioselective epoxidation of olefins to epoxides.281 Enantio-meric excess (ee) values up to 67% were obtained, althoughhigher ee values were associated with low conversion.

4.3. Artificial oxygen-activating enzymes by incorporationof metal cofactors into native scaffolds that do not containmetal ions

While native enzymes catalyze many reactions with high effi-ciency and selectivity, the scope of the reactions is still limitedin comparison to chemical catalysts, partially due to thelimitation of utilizing only physiological available metal ionssuch as iron and copper, and metal-containing cofactors suchas heme. To expand the scope of the reactions for more diversechemical transformations, chemical catalysts have been insertedin a non-metal-containing protein scaffolds. An initial example ofthis approach was given by Whitesides and coworkers, as early as1978, which utilized the extremely high affinity of protein avidinwith its substrate biotin and its variants (K B 10�15 M), to obtainan asymmetric hydrogenation catalyst. More specifically, a biotinconjugated variant of hydrogenation catalyst RhI–norbodiene wasconstructed and inserted into avidin.282 The tertiary structure ofthe enzyme avidin was expected to provide the chirality forenantioselective hydrogenation. Recently, Ward and coworkershave made significant progress in exploring, developing andrealizing the full potential of this strategy and much beyond(see Fig. 21a) in designing several biotin/streptavidin-basedmetalloenzymes to catalyze various enantioselective trans-formations.283–286 They showed that changes in the biotin-spacer-ligand moiety (see Fig. 21b), site-directed mutation onavidin, and metal ions had strong implications on the activityof the biotin-based catalyst. For example, an artificial metallo-enzyme for the reduction of prochiral imines was obtainedthrough the insertion of a biotinylated cyclopentadienyl rhodiumcomplex into a mutated streptavidin (Fig. 21a). Furthermore,an aminosulfonamide ruthenium complex (Fig. 21b), whenincorporated into avidin, oxidized secondary alcohols (usingtert-butylhydroperoxide as the oxidizing agent) with over 90%yield.287

Additionally, the authors also developed a vanadium-dependentartificial peroxidase for enantioselective sulfoxidation by inserting avanadyl ion into streptavidin.288 The vanadium–oxo complex [VO]2+

was inserted into the protein scaffold by using VOSO4 as avanadium source and the resulting hybrid protein catalyzedsulfoxidation of thioanisole giving 94% yield and 46% ee (R).EPR spectroscopy on the vanadium reconstituted protein yieldeda spectrum very similar to [VO(H2O)5]2+. This finding together



with docking experiments suggested that the interaction betweenvanadium and the protein was mainly dictated by hydrogenbonds in the second coordination sphere of the metal ion(Fig. 22). To improve the stereo-selectivity of the enzyme, ascreening on different prochiral sulfide substrates was performedwith enzyme displaying highest enantioselectivity for dialkyl andalkyl–aryl substrates (up to 93% ee). Interestingly, increasing thesteric bulk of the aromatic moiety of the sulfide led to an increasein selectivity showing the importance of proper substratepositioning in the active site for efficient catalysis.

Artificial metalloenzymes with peroxidase activity were alsodeveloped through the so-called ‘‘Trojan horse’’ strategy, byMahy and coworkers.289 They took advantage of the remarkableaffinity of a monoclonal antibody for its estradiol antigen(Kd = 9.5 � 10�10 M) to design artificial hemoproteins byinserting Fe- or Mn-porphyrin-estradiol conjugates into theanti-estradiol antibody (Fig. 23).289 These biocatalysts catalyzedselective oxidations, such as the enantioselective sulfoxidationof thioanisole by H2O2, with a 10% ee and the chemoselectiveepoxidation of styrene by potassium peroxymonosulfate(KHSO5). Differences in enantioselectivity were rationalized bydocking experiments, which suggested specific interactions of

the substrate with residues in the active site to be critical forenantioselectivity. Interestingly, the antibody still retains goodaffinity for its antigen, even when linked to such a bulkymolecule as a porphyrin, and interacts closely enough withthe catalytic center to influence its activity.

The same authors developed a different class of artificialhemoproteins, named ‘‘hemoabzymes’’, by raising monoclonalantibodies against metalloporphyrins and microperoxidases.290–295

In their first study, using FeIII-meso-tetrakis-ortho-carboxyphenylporphyrin (Fe-ToCPP) as the hapten, three monoclonal anti-porphyrin antibodies were found to recognize the porphyrin.Two of them, 13G10 and 14H7, bound to Fe-ToCPP withnanomolar Kd values and catalyzed the oxidation of ABTS byH2O2, exhibiting catalytic efficiencies at least 5-fold higher thanfree Fe-ToCPP.291–293 This result clearly highlighted the impor-tance of the protein matrix in modulating the activity of thecofactor. However, the catalytic efficiency of these enzymes wasfar lower than that of the native peroxidases. This finding wasattributed to the lack of an axial His ligand in catalytic iron. Toovercome this limitation, the authors used MP8 as the hapten(for details on MP8 see paragraph 2).290 Seven antibodiesrecognizing MP8 were produced, and the best of them, 3A3,was found to bind MP8 with a moderate Kd of 1 � 10�7 M.Despite the modest affinity, the 3A3–MP8 complex displayedgood peroxidase activity toward o-dianisidine characterized by akcat/KM value of 2 � 106 M�1 min�1. In addition, the 3A3–MP8complex catalyzed the regioselective nitration of phenol in thepresence of H2O2 with 2-nitrophenol as a preferred productover 4-nitrophenol.294 Moreover, the 3A3–MP8 complex wasshown to catalyze the oxidation of thioanisole by H2O2 in thepresence of 5% tert-butyl alcohol, with a 45% ee in favour of theR isomer.295

Chemical catalysts can also be incorporated into protein viaionic interactions. One such example comes from the Mahy lab,where Xylanase A (Xln10A, possessing a globally positive charge)was reconstituted with anionic porphyrins.296,297 Thus, Xln10A,which in its native form possesses no metal center, bound toFeIII-meso-tetrakis-p-carboxy-phenylporphyrin (Fe-TpCPP) and inthe presence of imidazole as a co-catalyst catalyzed the chemo-and stereoselective oxidation of thioanisole to sulfoxide, with85% yield and 40% ee.296 Replacement of iron with manganeseallowed for the selective oxidation of aromatic alkenes. Indeed,the Mn-based complex catalyzed the oxidation of para-methoxy-styrene by KHSO5 with a 16% yield and a very high enantio-selectivity (80% in favour of the R isomer).297

Fig. 21 Biotinylated complex inserted into streptavidin. (a) Crystal structureof a biotinylated cyclopentadienyl rhodium complex, inserted into theK121H streptavidine (PDB ID: 4gjs). (b) Molecular structure of the biotiny-lated aminosulfonamide ruthenium complex.

Fig. 22 Putative binding interaction between the vanadium oxo speciesand the streptavidin–biotin binding site. The interacting substrate is alsoshown for clarity. Adapted from ref. 288 Copyright 2008 AmericanChemical Society.

Fig. 23 Fe–porphyrin–estradiol conjugates. Reprinted with permissionfrom ref. 289. Copyright 2009 Elsevier.



The approach of constructing hybrid catalysts requires theselection of a protein capable of binding the catalyst and ofmodulating its activity in order to drive the targeted reaction.Given these requirements, several examples focused on the useof serum albumin as a protein scaffold, since the enzyme has aunique ability to bind a variety of hydrophobic molecules,including steroids, fatty acids and heme.298 A simple non-covalentadduct of albumin-conjugated manganese corroles was reported byGross and coworkers as a catalyst for asymmetric oxidations.299

The MnIII corrole reconstituted serum albumin catalyzed thesulfoxidation of asymmetric aryl-sulfide in the presence ofhydrogen peroxide with up to 74% ee. Further experimentsrevealed the direct involvement of the metal cofactor in thereaction mechanism and supported that the ee was due to thechiral environment surrounding the albumin-conjugated catalyst.Similarly, human serum albumin (HSA) was also functionalized byMnIII–salen complexes yielding MnIII–salen C HSA hybrid proteinsas Mn monooxygenase mimics (see Fig. 24 for MnIII–salen complexstructures).300 These hybrids have been proven to be very efficient(almost complete conversion of the substrate), stable (Kd values inthe micromolar range) and soluble in aqueous medium. Notably,comparison of the catalytic efficiency of the complexes and thehybrids shows that the polarity of the protein environment iscrucial for efficiency and selectivity. In fact, the hybrids selectivelycatalyzed the oxidation of thioanisole, by sodium hypochlorite, tothe corresponding sulfoxide, without further oxidation to sulfone.

All the examples discussed above were built upon visualinspection of proteins and their physico-chemical properties toinsert a chemical catalyst into them. As an alternate strategy,Ward and coworkers developed a computer search algorithm,called ‘‘Search for Three-dimensional Atom Motifs in ProteinStructure’’ (STAMPS), which helps identify protein structureswith a given topological motif similar to the functional motif ina reference protein, through a systematic search in the proteindatabank (PDB).301,302 Using this approach, the authors demon-strated that it is possible, by the in silico study, to create anartificial metallo-peroxidase upon addition of a metal cofactorto a non-metal-containing protein that harbors a potential metal-binding site. A search within the PDB allowed identifyingpre-organized two histidine and one carboxylate triads in proteinsthat appeared to be suitable for metal ion coordination.303 Severalscaffolds bearing an HHD/E motif, located within a pocketpredisposed to bind metals, were selected. The search wasfurther expanded to include HHN/Q motifs, revealing six addi-tional potential metal binders following a single point mutation.Out of the thirteen cloned proteins, six were overexpressed inE. coli. One of these proteins, 6-phosphogluconolactonase,

bearing a N131D mutation (6-PGLac), bound CuII and showedperoxidase activity toward o-dianisidine. Structural insightsinto the metalloenzyme activity were gained by X-ray crystallo-graphy that confirmed copper ion to be coordinated by two Hisresidues. This strategy looks very promising since it is possibleto generate novel functionality within a protein scaffold, andcan be valuable for mechanistic enzymology and evolutionaryanalysis of enzymatic activity.

Another successful example of redesigning protein activity isthe work carried out by Sheldon and coworkers, who developedsemi-synthetic peroxidase through the incorporation of vanadateions into acid phosphatases and phytases.304–307 Towards thisgoal, the authors exploited the structural similarity betweenvanadium haloperoxidases and the acid phosphatases. Tobegin with, vanadate (VO4

3�) and phosphate ions (PO43�) are

very similar in their structures. In addition, in the apo-form,vanadium haloperoxidases (which contain a vanadate ion)exhibit phosphatase-like activity, while vanadate ions arepotent inhibitors of the phytase and sulfatase activity. Thus,vanadium haloperoxidases and phosphatases (which bind andhydroxylate phosphate) were considered to have similar activesites (Fig. 25).308 Based on these structural and functionalsimilarities, the authors incorporated vanadate ions into phos-phatases to produce novel, semi-synthetic peroxidases. Theresulting vanadium phosphatase catalyzed the enantioselectiveoxidation of several sulfides, with low ee. Furthermore, thesemi-synthetic peroxidase was found to be more stable atrelatively high H2O2 concentrations (as compared to vanadiumhaloperoxidase) displaying catalytic activity approaching that ofheme peroxidases (see Table 3).

Another method to incorporate metal cofactors artificiallyinto protein scaffolds is by adding metal-coordinating unnaturalamino acids (UAAs).77,309 UAAs are now routinely being incorpo-rated into peptides and proteins to enable functions beyond thoseaccessible using natural amino acids.310–314 UAAs have been used

Fig. 24 Mn salen complexes inserted into HSA.

Fig. 25 Active site crystal structure of (a) vanadium chloroperoxidase(PDB ID: 3w36) and (b) phytase (PDB ID: 4aro).

Table 3 Comparison of the catalytic properties of three different perox-idases based on the oxidation of thioanisolea

Heme CPOC. fumago

Phytase/VO43� VBPO

A. nodosum

ee (%) 99 (R) 68 (S) 83 (R)TOF (min�1) 900 5.5 0.78TTN (�) 108 000 25 000 750




to enable new modes of metal coordination or covalent attachment,which has led to the construction of several new artificial metal-loenzymes and metallopeptides not possible using natural aminoacids. In particular, Lewis and coworkers have recently used proteinscaffolds, selectively modified with the UAA residue p-azido-L-phenylalanine (Az), to covalently link catalytically active alkyne-substituted metal complexes, via strain-promoted azide–alkynecycloaddition.315 For example, an alkyne-substituted dirhodiumcatalyst, when incorporated into prolyl oligopeptidase (POP),performed highly selective (92% ee) olefin cyclopropanation.316

Even though this strategy has not yet been used for developingoxygen-activating metalloenzymes, it deserves great attention sinceit can be used, in principle, to conjugate any desired metal complexto various scaffolds, targeting different functions.

5. Future targets for metalloproteindesign

Many oxygen-activating metalloenzymes have recently beendiscovered and characterized, offering exciting opportunitiesfor the design and engineering of novel artificial enzymes. Inthis section, we give few examples of iron and copper-basedoxygen-activating enzymes, which represent important futuretargets for metalloprotein design. Reproducing their intriguingfeatures in model systems will not only expand the repertoire ofartificial metalloenzymes but it will also be valuable in biotechno-logical applications.

5.1. a-Ketoglutarate (aKG) dependent dioxygenaseand halogenase

They are a class of mononuclear non-heme iron-dependentdioxygenases that require aKG as a cosubstrate to performcatalysis. In fact, these enzymes couple the oxidative decarboxyl-ation of the cofactor aKG to carbon dioxide and succinate to theoxidative transformations of a variety of substrates. Crystallo-graphic data from several members of this class reveal acommon structural arrangement of the active site that consistsof a FeII metal center, facially coordinated by two histidines andone carboxylate ligand, from either a glutamate or aspartateresidue.5 This ‘‘facial triad motif’’ leaves empty three remainingsites, which are occupied by weakly bound solvent molecules inthe resting-state enzyme, and thus are available for binding ofadditional exogenous ligands, as the aKG cofactor or substrates.This flexibility in the coordination environment at the metalaccounts for the observed diversity in catalyzed oxidativetransformations.8

Among aKG-dependent dioxygenases, taurine/2-oxoglutyratedioxygenase (TauD) catalyzes the hydroxylation of taurine,enabling E. coli to use the aliphatic sulfonate taurine as asulfur source during periods of sulfate starvation.317 The non-heme iron in TauD activates the oxygen molecule, cleaving theO–O bond to obtain a high-valent iron oxo intermediate, whichis then able to abstract an H radical from the nearby C–H bondto perform hydroxylation. Analogous to TauD are another set ofnon-heme iron dependent enzymes, halogenases, that catalyze

the site-selective chlorination/bromination of saturated hydro-carbon (specifically the C-4 position of amino acid Thr).318 Thecrystal structure of SyrB2 halogenase in the resting state revealsthat the non-heme iron exhibits a facial triad geometry similarto TauD, except that the Glu/Asp ligand of TauD is replaced by ahalogenide (Cl� or Br�).319 Furthermore, the mechanism ofoxygen/C–H bond activation and O–O bond cleavage in SyrB2 isalso similar to that of TauD except that the halogenase veryselectively halogenates the C–H bond with almost no hydroxyl-ated product.320 Studies on the structure–function relationship ofSyrB2 have revealed that the hydrogen-bonding to the ferrylintermediate, as well as the positioning of the substrate, playsan important role in controlling the chemo- and regio-selectivityof this reaction. However, exact details of this control are notunderstood especially because the SyrB2 is dependent on aconjugate enzyme SyrB1.321–323 These two examples highlightonce again that similar active sites are able to perform verydifferent functions, depending on the protein environment.Thus, they are interesting candidates for metalloprotein design,as controlling secondary coordination sphere interactions andsubstrate positioning is nowadays feasible using the tools ofprotein design and engineering.

5.2. Polysaccharide monooxygenases

An exciting target for future copper–protein design and engi-neering is the polysaccharide monooxygenases (PMOs), alsoknown as lytic PMOs (LPMOs), which utilize molecular oxygento cleave glycosidic bonds.18–21 The interest in this class ofenzymes is growing considerably, since they enhance thedepolymerization of recalcitrant polysaccharides, such as cellulose,chitin and diverse marine polysaccharides, by hydrolytic cleavage.In this respect, PMOs hold commercial potential in theenhancement of biomass degradation, and could find applicationin industrial biofuel production. Several structural, spectroscopicand activity studies have just started to shed light on the featuresof these powerful enzymes.20,22 PMOs are now known to becopper-dependent oxygenases with unusual active sites, since theyperform O2 activation using only a single Cu center. The PMOactive site contains a mononuclear type II copper center coordi-nated by three equatorial nitrogen ligands in a histidine bracemotif. Solomon and coworkers have recently determined thecoordination properties of the PMO from T. aurantiacus insolution.22 Using a combination of spectroscopic and computa-tional analyses, they found a different geometry for CuII and CuI:a four-coordinate tetragonal geometry in the oxidized state and athree-coordinate T-shaped structure in the reduced state. Threeprotein-derived nitrogen ligands coordinate copper in both redoxstates, whereas a labile hydroxide ligand is lost upon reduction.The unique protein structure favors the formation of the T-shapedCuI site, providing an open coordination position for strong O2

binding with very little reorganization energy, and enabling thethermodynamically difficult one-electron reduction of O2 byCuII-superoxide formation. This work represents an importantstep toward elucidating the mechanism by which mononuclearCu oxygenases activate the inert O2 molecule by one-electronreduction for subsequent degradation of polysaccharides.



Reproducing this mechanism of O2 reduction by a singlecopper center in either de novo designed or engineered proteinscaffolds will be challenging for protein designers, but will alsoaid in the development of more and more efficient enzymes.

5.3. Methane monoxygenases (MMOs)

MMOs are key enzymes in the aerobic bacterial degradation ofhydrocarbons. Their catalytic chemistry is extraordinary, sincethey catalyze the activation of the extremely strong C–H bondof methane to methanol, under ambient conditions, withoutover-oxidation.324–327 There are two distinct types of MMOs, aniron-dependent, soluble cytosolic protein (sMMO), and a copper-dependent membrane protein, particulate MMO (pMMO). Eventhough the two enzymes perform the same transformation, theirstructures, active sites, and chemical mechanism are completelydifferent. A huge amount of structural and functional data isavailable for the sMMO, and in particular for its diiron activecenter. In contrast, structural features responsible for the activityof copper-containing pMMO and its catalytic mechanism remainto be fully elucidated.324–328 The protein models of MMOs mayhelp in understanding how the two different MMO metal centersefficiently accomplish this challenging chemistry under physio-logical conditions.

6. Conclusions and futureperspectives

The field of design and engineering of artificial metalloenzymeshas progressed spectacularly within the last decade. What startedas minimal structural mimics that have no activity has evolved intofunctional metalloenzymes achieving rates close to those of nativeenzymes. Much of the progress has been made as a result of recentdevelopment in computational and molecular biology. At the sametime, advances in structural biology, in terms of obtainingcrystallographic and NMR structures, also help in better character-ization of the resulting designed enzymes to further improve them.In this review, we have used design of oxygen-activating metallo-enzymes as examples to illustrate progress made so far and tohighlight approaches employed to achieve this progress. Signi-ficant advances have been made in designing many oxygen-activating metalloenzymes by miniaturization, by de novo designand by protein redesign using native protein scaffolds. For allthree practices, both rational designs using computer modelingand directed evolution or combinatorial selection have beenemployed. Suitable practices or approaches to be used dependon: (i) the level of the understanding of the enzyme one aims todesign; (ii) the reaction one targets to achieve; (iii) the technicaldifficulty inherent to the selected design. For instance, proteinminiaturization is preferred if the geometric parameters of themetal cofactors are well defined, with minimal help from theprotein scaffolds to maintain their stability and integrity.37,113

Different types of heme cofactors are excellent examples andthat is why miniaturization of heme-enzymes has enjoyed mostsuccess in this practice.85,113 When the geometric parametersof the metal-binding sites require substantial participation of

protein scaffolds to define and maintain, such as diiron centers,one needs to consider applying either design using de novoscaffolds, or redesign using native scaffolds. Protein designusing de novo scaffolds is an ultimate goal in engineeringenzymes, as it is a testimony of complete understanding of theproteins. Only limited de novo designed scaffolds are available andmost success in de novo design of functional metalloproteins hasbeen reached using helical bundles as scaffolds.35,38,39,61 They areso stable to tolerate multiple mutations without disruption of theglobal folding. This is allowing one to explore how changes tothe first- and second-shell ligands, as well as residues lining thesubstrate binding pocket, affect the reactivity and catalytic pro-perties of the metal site. The design of metal sites that are nativelyinserted in helical bundles is relatively easy, thus allowing one tounravel each specific contribution to function. This also enablesone to clearly identify which feature could be responsible forswitching towards alternative reactivity. In this perspective,various examples show how the initial designed function canbe finely tuned, or even reprogrammed.162,165,173 Moreover, it ispossible to engineer metal sites that are not natively found inhelical bundles, by taking advantage of intrinsic symmetry ofthe natural coordination environment, which can be fitted wellin a de novo designed coiled coil. An outstanding example wasreported by Pecoraro and coworkers, who engineered thethreefold symmetric site of carbonic anhydrase, an all betaenzyme, into a three-stranded coiled coil.58 Finally, it should benoted that the field is expanding, and other than a-folds arebeing investigated, in an attempt to fully de novo design proteinstructures beyond those found in Nature.329

For metal-binding sites that are not easily designable in helicalbundles, design using native scaffolds becomes advantageous. Inaddition, with the combination of gains in our knowledge andexperience in protein design together with recent advances inmodern computational biology and biophysical characterizationtechniques, rational design has enjoyed much success recently.However, there are still a number of gaps in our knowledge andcomputational methods, especially when we wish to designenzymes with very high activity. In this case, directed evolutionsbecome very useful and can complement rational design well. Infact, some of the most successful examples of artificial enzymesare results of using the combined approach.202,330,331

Most reported designs of metalloenzymes aim to gain deeperinsights into how metalloproteins are assembled. Given muchsmaller and more well-defined structures, these designedproteins have provided deeper insights into the structure,mechanism and function of the ‘‘target’’ protein. For example,in the design of N-oxygenase, DeGrado and coworkers contri-buted to clarifying the role of the third histidine in the primarycoordination sphere of a diiron center in the N-oxygenationreaction.165 Moreover, designing the CcO active site in myoglobingave considerable inputs into the role of different structuralfeatures like conserved tyrosines and different heme types inCcOs.25,228,234 In some exceptional cases, a number of artificialmetalloenzymes have been engineered to have an impact inchemical and biotechnological applications. For example, CYPmutants developed by Arnold and coworkers can now oxidize



ethane to ethanol (an industrial raw material that is typicallyproduced via high-energy thermochemical methods) with highselectivity, without formation of over-oxidized products.206 ThisCYP mutant along with others is currently being utilized inbiotech startup companies to obtain fuel/raw materials fromcellulose and lignin.

While the progress made so far is quite exciting, much morework is required to reach the full potential of the field. Forexample, most artificial metalloenzymes designed for oxygen-activation reviewed here use highly reactive and unstable H2O2

as the sacrificial oxidant. For further advancement of this fieldand to be able to use these designed metalloenzymes for wholecell biotransformation suitable for fermentation, metallo-enzymes using bioavailable and inexpensive O2 as the oxidantneed to be developed. This is especially important if we wish toscale up the reactions for industrial use. The issue in usingoxygen is that the O–O bond is difficult to break efficiently, andthus lessons should be learnt from native enzymes like CYPs332

to design oxygen-utilizing artificial metalloenzymes.For non-heme metal-binding sites, the metal-binding affinity

of the designed site is normally much weaker than those of nativeenzymes making the metal-binding site much less selective thanthose of native enzymes. This renders the designed metallo-enzyme more difficult to be characterized and the enzymaticassays hard to be performed at low concentrations. Suchlimitations could be overcome through site-specific incorporationof metal-chelating unnatural amino acids by means of recentgenetic code expansion techniques. This strategy can at thesame time recapitulate the functions of metalloproteins bearinguncommon metal-binding sites and expand the native functionsresulting in molecular systems with unprecedented efficiencyand selectivity.314

More importantly, with a few exceptions,58,59,113,162,228 mostdesigned metalloenzymes have very low activity and turnovernumbers. A main contributing factor to both issues is thenon-covalent secondary sphere interactions around the primarycoordination sphere that influence both the metal-bindingaffinity and enzymatic efficiency. While more recent reportshave begun to address this issue,113,162,165,170,238 more attentionneeds to be paid in designing non-covalent secondary sphereinteractions.

Furthermore, only a very small number of oxygen-activatingenzymes, described in this review, perform in whole cells thatare amenable to producing products by fermentation. To thischallenge, more emphasis should be given into using moderateoxidants like oxygen and chlorite for catalytic biotransformation.Moreover, light energy can be stored in the form of chemicalenergy by converting light into energy for oxidation reaction. Onesuch recent example is by Lewis and coworkers, who utilizedthe acridinium cofactor embedded in a protein to performsulfoxidation.333 Ru–bipy conjugated heme has been incorpo-rated into CYPs in attempts to perform light based C–H bondoxidation.334 While this is an excellent idea, the photo-catalystsdesigned until now are not very selective and display lowquantum efficiency. Thus, more impetus should be providedto develop light-based oxidation catalysts.

Overall, the metalloenzyme design has allowed chemists,biochemists and chemical engineers to answer challengingquestions about metal biochemistry, the importance of theprotein matrix, and ultimately be able to design new metallo-proteins capable of performing desired functions not necessarilyin the repertoire of biology. The examples herein discussed makesignificant progress toward these goals.

Acknowledgements

We wish to thank all coworkers and collegues for their contri-butions to the results described in this review, as well asFabrizia Sibillo for help with editing the manuscript. The workfrom the authors’ groups reported herein has been supportedby US National Institutes of Health (GM062211), US NationalScience Foundation (CHE 14-13328), the European Union (EU)(Cost Action CM1003 – Biological Oxidation Reactions:Mechanisms and Design of New Catalysts) and the ScientificResearch Department of Campania Region (BIP Project, PORFESR 2007/2013, grant number B25C13000290007, and the STRAINProject, POR FSE 2007/2013, grant number B25B0900000000 for apostdoctoral fellowship to M. C.).

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Chem Soc RevAmbika Bhagi-Damodaran Ambika Bhagi-Damodaran was born in Moradabad, India. She received...

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Transcript of Chem Soc RevAmbika Bhagi-Damodaran Ambika Bhagi-Damodaran was born in Moradabad, India. She received...