molecules

Review Rational Design of Artificial Metalloproteins and Metalloenzymes with Metal Clusters

Ying-Wu Lin 1,2,3

1 School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China; [email protected] or [email protected]; Tel.: +86-734-8578079 2 Laboratory of Protein Structure and Function, University of South China, Hengyang 421001, China 3 Hunan Key Laboratory for the Design and Application of Actinide Complexes, University of South China, Hengyang 421001, China



Received: 21 June 2019; Accepted: 26 July 2019; Published: 29 July 2019


Abstract: Metalloproteins and metalloenzymes play important roles in biological systems by using the limited metal ions, complexes, and clusters that are associated with the protein matrix. The design of artificial metalloproteins and metalloenzymes not only reveals the structure and function relationship of natural proteins, but also enables the synthesis of artificial proteins and enzymes with improved properties and functions. Acknowledging the progress in rational design from single to multiple active sites, this review focuses on recent achievements in the design of artificial metalloproteins and metalloenzymes with metal clusters, including zinc clusters, cadmium clusters, iron–sulfur clusters, and copper–sulfur clusters, as well as noble metal clusters and others. These metal clusters were designed in both native and de novo protein scaffolds for structural roles, electron transfer, or catalysis. Some synthetic metal clusters as functional models of native enzymes are also discussed. These achievements provide valuable insights for deep understanding of the natural proteins and enzymes, and practical clues for the further design of artificial enzymes with functions comparable or even beyond those of natural counterparts.

Keywords: metalloproteins; metalloenzymes; protein design; metalclusters; synthetic models

1. Introduction Metalloproteins and metalloenzymes play important roles in biological systems, including electron transfer, O2 binding and delivery, and catalysis, etc. [1–11]. Despite the functional diversity, the cofactor or prosthetic group of native metalloproteins/metalloenzymes are made of about 14 metal ions, and several types of metal complexes (such as heme) or metal clusters (such as iron–sulfur clusters) [4,5,10], which may limit the functionalities of native metalloenzymes. To overcome these limitations, it is desirable to rationally design artificial metalloproteins and metalloenzymes, which have received great attention for several decades, and achieved significant progresses, such as for artificial oxidases and reductases, etc. [12–29]. These progresses not only reveal the key structural elements responsible for the activity of native enzymes, but also endows the ability to create artificial enzymes with improved properties and functions. In the design of artificial metalloproteins and metalloenzymes, many strategies have been developed, such as redesign of the active metal site by fine-tuning the cofactor–protein interactions through site/loop-directed mutagenesis [30–32], the design of new metal-binding sites [33–38], the incorporation of unnatural amino acids and non-native cofactors into native or de novo protein scaffolds [20,25,39–41], and the directed evolution of metalloenzymes [23,42], as well as the use of post-translational modifications (PTMs) [43–50]. Other materials such as nanoparticles and hydrogels have also been used for the design of enzyme mimics [51–53]. Moreover, these designs have been

Molecules 2019, 24, 2743; doi:10.3390/molecules24152743 www.mdpi.com/journal/molecules Molecules 2019, 24, 2743 2 of 18 Molecules 2019, 24, x FOR PEER REVIEW 2 of 18 constructionachieved for metalloproteinsof the mononuclear and metalloenzymes site via metal with substitution single to multiple or incorporation, active sites bythe construction design of homodinuclearof the mononuclear or heterodinuclear site via metal si substitutiontes and the orreconstitution incorporation, of themetal design complexes, of homodinuclear as well as the or designheterodinuclear of dual and sites multiple and the reconstitutionactive sites in ofsingle, metal dimeric complexes, proteins, as well and as protein the design oligomers of dual and polymersmultiple active [15]. The sites progress in single, in dimeric this field proteins, has also and been protein well oligomersreviewed andvery polymers recently [[21–29].15]. The progress in thisComparatively, field has also beenthere well has reviewed been less very progress recently in [21the–29 design]. of artificial metalloproteins and metalloenzymesComparatively, with there metal has clusters, been lessespecially progress for inthose the formed design by of artificialmultiple metalloproteinsmetal ions. In 2016, and Fehlmetalloenzymes and Davis critically with metal reviewed clusters, the especially progress forin the those design formed of heterogeneous by multiple metal catalysis ions. by In using 2016, proteinsFehl and Davisor polypeptides critically reviewed as templates the progress for synthetic in the design metalloclusters, of heterogeneous including catalysis iron–sulfur by using clusters, proteins di-ironor polypeptides clusters, asand templates nickel–iron for synthetic clusters metalloclusters, [54]. In 2018, Ueno including et al iron–sulfur. highlighted clusters, the research di-iron clusters,on the functionalizationand nickel–iron clusters of protein [54]. Incrystals 2018, Uenowith etmetal al. highlightedions, complexes, the research and nanoparticles on the functionalization [55]. In this of review,protein crystalswe summarize with metal the ions, very complexes, recent progress and nanoparticles in the rational [55]. Indesign this review, of artificial we summarize metalloproteins the very andrecent metalloenzymes progress in the rationalby focusing design on of the artificial design metalloproteins of multi-metal and cluste metalloenzymesrs, as well as by some focusing synthetic on the metaldesign clusters of multi-metal as functional clusters, models as well of as native some en syntheticzymes. metalThe mapping clusters asof functionalthe diverse models metal ofclusters native thatenzymes. have Thebeen mapping designed of theto diversedate in metalthe periodic clusters thattable have is shown been designed in Figure to date1. These in the achievements periodic table provideis shown not in Figureonly valuable1. These insights achievements into the provide structure–function not only valuable relationship insights into of native the structure–function metalloproteins andrelationship metalloenzymes, of native metalloproteinsbut also practical and clues metalloenzymes, for creating more but alsoadvanced practical artificial clues formetalloenzymes, creating more whichadvanced will artificial ultimately metalloenzymes, stimulate the which growth will and ultimately expansion stimulate of this the field. growth and expansion of this field.

Figure 1. ArtificialArtificial metalloproteins and and metalloenzymes with with diverse diverse metal metal clusters shown in the periodic table. 2. Artificial Metalloproteins with Metal Clusters for Structural Roles 2. Artificial Metalloproteins with Metal Clusters for Structural Roles 2.1. Zinc Clusters 2.1. Zinc Clusters Zinc ions play crucial roles in both protein scaffolds and protein–protein interfaces, acting as catalyticZinc sites ions or play supporting crucial roles quaternary in both protein protein structures scaffolds [56 and]. Tezcan protein–protei et al. showedn interfaces, that by designacting as of catalytic sites or supporting quaternary protein structures [56]. Tezcan et al. showed that by design azinc-binding site on the protein surface, such as on c-type cytochrome b562 (Cytcb562) with a covalently ofattached azinc-binding heme, the site protein on the assembles protein insurface, ordered such structures as on as c-type dictated cytochrome by the binding b562 (Cyt of Zncb2562+ )ions with [57 a]. covalently attached heme, the protein assembles in ordered structures as dictated by the binding of Both structural zinc sites (3-His-1-Asp) and catalytic zinc sites (2-His-1-Glu-1-H2O) can be designed in Zn2+ ions [57]. Both structural zinc sites (3-His-1-Asp) and catalytic zinc sites (2-His-1-Glu-1-H2O) Cytcb562 assembly, which confer a stable and active artificial hydrolase both in vitro and in vivo [58]. can be designed in Cytcb562 assembly, which confer a stable and active artificial hydrolase both in Note that Cytcb562 is a four-helix bundle protein, and can form a dimer by domain-swapping, wherethe vitroN-terminal and in twovivo helices [58]. Note of one that protomer Cytcb562 mayis a four-helix interact with bundle the C-terminalprotein, and two can helices form ofa dimer the other by domain-swapping, wherethe N-terminal two helices of one protomer may interact with the protomer. Moreover, Hirota et al. observed that three domain-swapped Cytcb562 dimers can further C-terminal two helices of the other protomer. Moreover,2+ Hirota et2 al. observed that three form a unique nanocage, witha novel Zn–SO4 cluster (15 Zn and 7 SO4 − ions) inside the cavity, as domain-swappedshown in the X-ray Cyt crystalcb562 structuredimers can (Figure further2a) form [ 59]. Ina unique addition nanocage, to the coordination witha novel between Zn–SO Zn4 cluster2+ and (15 Zn2 2+ and 7 SO422+− ions) inside the cavity, as shown in the X-ray crystal structure (Figure 2a) [59]. SO4 − ions, the Zn ions in the Zn–SO4 cluster were also coordinated by the amino acid side chains of 2+ 2− 2+ Inthe addition dimers, whichto the coordination stabilized the between cage structure. Zn and Additional SO4 ions, stabilization the Zn ions eff inects the were Zn–SO contributed4 cluster were from alsoa hinge coordinated loop thatconnected by the amino two acid four-helix side chains bundle of units. the dimers, which stabilized the cage structure. Additional stabilization effects were contributed from a hinge loop thatconnected two four-helix bundle units.

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As an alternative to native proteins, de novodesigned proteins such as helical bundles provide idealAs scaffolds an alternative for the todesign native ofproteins, artificial demetalloproteins novodesigned by proteins the incorporation such as helical of metal bundles ions, provide metal idealcomplexes, scaffolds or formetal the clusters design of[36,60]. artificial For metalloproteins example, Pecoraro by the et incorporation al. designed ofa metalZn2+-binding ions, metal site 2+ complexes,(3-His-1-H2O) or inthree-helical metal clusters bundles, [36,60]. Forwhich example, confers Pecoraro an impressive et al. hydrolase designed aactivity Zn -binding toward sitethe (3-His-1-Hhydration of2O) CO inthree-helical2 with an efficiency bundles, comparable which confers to that an of impressivenative carbonic hydrolase anhydrases activity [61]. toward DeGrado the hydrationet al. designed of CO a2 dinuclearwith an e ffizincciency site comparablein four-helical to thatbundles, of native with carbonic two Zn2+ anhydrases ions bridged [61 by]. DeGrado two Glu etresidues al. designed and coordinated a dinuclear by zinc additional site in four-helical His and bundles,Glu residues. with twoWith Zn a2 +suitableions bridged substrate-binding by two Glu residuespocket, this and coordinatedde novo protein by additional was able His to and stabilize Glu residues. the radical With semiquinone a suitable substrate-binding form of catechols pocket, for thisweeks, de novowhich protein is otherwise was able unstable to stabilize in aqueous the radical solution semiquinone [62]. Recently, form of Lombardi catechols foret al. weeks, designed which a istetranuclear otherwise zinc unstable cluster in within aqueous four-helical solution [ 62bundles]. Recently, (named Lombardi 4D/EH1/2), et al. which designed consist a tetranuclearof four Zn2+ zincions clusterand four within carboxyl four-helical oxygens bundles from Asp/Glu (named 4D(Figure/EH1 /2b)2), which[63]. Additional consist of fourligands Zn2 +wereions provided and four carboxylby His residues, oxygens which from Aspwere/Glu further (Figure stabilized2b) [ 63 ].by Additional second-shell ligands and third-shell were provided interactions, by His residues,forming whicha fully wereconnected further H-bond stabilized network. by second-shell By optimizing and third-shell the amino interactions, acid sequence, forming the a fullypeptide connected can be H-bonddesigned network. to form a By tetramer optimizing in aqueous the amino solution acid in sequence, the absence the peptideof metal can ions, be which designed subsequently to form a tetramerbinds Zn2+ in ions aqueous and forms solution a tetranuclear in the absence cluster, of metal suggesti ions,ng which an impact subsequently of the designed binds Zn geometry2+ ions and to formsthe metal a tetranuclear cluster [64]. cluster, suggesting an impact of the designed geometry to the metal cluster [64].

Figure 2.2. (a(a)) Cage Cage structure structure of threeof three domain-swapped domain-swapped Cytcb Cyt562 dimerscb562 dimers (PDB code(PDB 5AWI), code 5AWI), and a close-up and a 2+ 2+ 2 2− viewclose-up of theview Zn of theand Zn SO 4and− ions SO4 in ions the in internal the internal cavity. cavity. Reprinted Reprinted with permissionwith permission from from Ref. [Ref.59], Copyright[59], Copyright 2016 The2016 Royal The SocietyRoyal Society of Chemistry; of Chemistry; (b) Crystal (b structure) Crystal ofstructure 4DH1 (PDB of 4DH1 code 5WLL),(PDB code and a5WLL), close-up and view a close-up of the designed view of tetranuclearthe designed zinc tetran cluster.uclear Reprinted zinc cluster. with Re permissionprinted with from permission Ref. [63], Copyrightfrom Ref. [63], 2018 Copyright American 2018 Chemical American Society. Chemical Society.

2.2. Cadmium Clusters 2.2. Cadmium Clusters

Similar to a tetranuclear copper cluster (Cu4S4) that can be designed within a four-helical Similar to a tetranuclear copper cluster (Cu4S4) that can be designed within a four-helical bundle [65,66], a tetranuclear cadmium cluster can also be designed, such as in the interior of a bundle [65,66], a tetranuclear cadmium cluster can also be designed, such as in the interior of a three-helical bundle, by using a metal-binding motif of CXXCE [67]. As shown by X-ray crystallography three-helical bundle, by using a metal-binding motif of CXXCE [67]. As shown by X-ray (Figure3a), the tetra-Cd 2+ clusteris a tetrahedral adamantane-like cluster, with four Cd2+ ions bridged crystallography (Figure 3a), the tetra-Cd2+ clusteris a tetrahedral adamantane-like cluster, with four by six Cys residues and coordinated by three Glu residues, as well as an additional water molecule, i.e., Cd2+ ions bridged by six Cys residues and coordinated by three Glu residues, as well as an [Cd4(µ2-S Cys)6(O2C Glu)3(H2O)], resulting in a very stable de novo designed metalloprotein. additional· water molecule,· i.e., [Cd4(μ2-S·Cys)6(O2C·Glu)3(H2O)], resulting in a very stable de novo designedFerritin metalloprotein. (Fr) is a cage-like protein for iron storage, which is formed by the assembly of 24 subunitsFerritin [(Fr)68]. is As a acage-like result, theprotein structure for iron is highly storage, symmetrical, which is formed with twofold,by the assembly threefold, of and 24 fourfoldsubunits symmetry[68]. As a result, axes. the The structure threefold is highly axis channel symmetrical, is hydrophilic, with twofold, and canthreefold, be used and for fourfold metal penetration.symmetry axes. Diff erently,The threefold the fourfold axis channel axis channel is hydrophilic, has a hydrophobic and can microenvironment.be used for metal penetration. Inspired by theDifferently, use of de the novofour-helical fourfold axis bundleschannel forhas the a hydrophobic design of metal-binding microenvironment. sites [69], Inspired Ueno et al.by introducedthe use of twode novofour-helical Cys residues (L161C bundles/L165C for or the L168C design/L69C of mutation)metal-binding at the sites fourfold [69],axis Ueno channel et al. ofintroduced apo-Fr, which two 3+ leadsCys residues to the formation (L161C/L165C of four or orL168C/L69C eight binding mutation) sites for at Cd the fourfoldions, as axis revealed channel by theof apo-Fr, X-ray crystalwhich structures [70]. For example, the X-ray structure of apo-L161C/L165C-Fr showed that a Cd -cluster is leads to the formation of four or eight binding sites for Cd3+ ions, as revealed by the X-ray8 crystal located at the fourfold axis channel (Figure3b), which is coordinated by Cys161 and /or Cys165 with a structures [70]. For example, the X-ray structure of apo-L161C/L165C-Fr showed that a Cd8-cluster distanceis located of at 2.4 the to fourfold 2.5 Å, as wellaxis aschannel water (Figure molecules 3b), (2.4 which Å). The is coordinated Cd–Cd distance by Cys161 was found and/or to be Cys165 3.4 Å, whichwith a agreesdistance with of 2.4 thoseof to 2.5 the Å, Cd–Sas well cluster as water in naturalmolecules metallothionein (2.4 Å). The Cd–Cd (3.4 to distance 4.4 Å), suggesting was found the to possiblebe 3.4 Å, formation which agrees of a metal–metal with thoseof bond, the althoughCd–S cluster the occupanciesin natural metallothionein of both Cd ions (3.4 were to less 4.4 than Å), onesuggesting (0.6 and the 0.2, possible respectively). formation This of study a metal–metal suggests thatbond, not although only the the threefold occupancies axis channel, of both butCd ions also were less than one (0.6 and 0.2, respectively). This study suggests that not only the threefold axis

MoleculesMolecules 20192019,, 2424,, x 2743 FOR PEER REVIEW 4 4of of 18 18 channel, but also thefourfold axis channelof Fr can be used for design of metal clusters, with the coordinationthefourfold axis structure channelof controlled Fr can beby usedthe assembly for design interfaces of metal of clusters, protein with cages. the coordination structure controlledIn addition by the to assembly the Cd–sulfur interfaces cluster, of protein cadmium cages. can form clusters with inorganic ions such as chlorideIn addition ions. Rose to the et Cd–sulfural. found that cluster, when cadmium crystallized, can form the clusters protein with augmenter inorganic of ions liver such regeneration as chloride containingions. Rose eta al.14-residue found that hexahistidine when crystallized, purification the protein tag (termed augmenter hsALR) of liver in regeneration the presence containing of a high a concentration14-residue hexahistidine of CdCl2 purification(50 mM), the tag (termedprotein hsALR)formed ina thetetramer presence composed of a high concentrationof two homodimers, of CdCl2 which(50 mM), was the bridged protein by formed a novel a tetramer Cd2Cl4O composed6 cluster with of two coordination homodimers, by whichtwo Asp was residues, bridged as by well a novel as twoCd2 Clwater4O6 clustermolecules with (Figure coordination 3c) [71]. by Moreover, two Asp residues, the formation as well asof twoa cadmium water molecules chloride (Figurecluster3 canc) [ 71be]. rationallyMoreover, thedesigned formation by ofusag a cadmiume of the chlorideprotein clusterinterface. can beAs rationally designed designed by Voet by et usage al., ofan the artificial protein βinterface.-propeller As protein designed named by Voet Pizza et al.,forms an artificiala trimerβ with-propeller threefold protein symmetry. named PizzaBy the forms further a trimer design with of metal-bindingthreefold symmetry. sites Byusing the furtherHis as designligands, of metal-bindingthe new version sites of using nvPizza2-S61H58 His as ligands, thewas new found version to accumulateof nvPizza2-S61H58 cadmium was and found chloride to accumulate ions by cadmiumdimerization and chlorideof the trimer, ions by forming dimerization a novel of the Cd trimer,7Cl12 clusterforming at a the novel threefold Cd7Cl12 symmetriccluster at the axis threefold (Figure symmetric3d) [72]. The axis cadmium (Figure3d) ions [ 72 ].were The coordinated cadmium ions by were the Hiscoordinated residues byfrom the each His residuessubunit, fromas well each as subunit,chloride as ions, well resulting as chloride in a ions, lattice resulting that is innearly a lattice identical that is tonearly that identicalof crystalline to that cadmium of crystalline chloride. cadmium This study chloride. suggests This studythat symmetric suggests that proteins symmetric may proteinsbe used formay the be biomineralization used for the biomineralization of nanocrystals of nanocrystalswith useful properties. with useful properties.

FigureFigure 3. 3. ((aa)) X-ray X-ray crystal crystal structure structure of of a a tetranuclear tetranuclear Cd Cd–thiolate–thiolate cluster cluster (PDB (PDB code code 4G1A), 4G1A), and and the the coordinationcoordination structure. structure. Reprinte Reprintedd with with permission permission from from Ref. Ref. [67], [67], Copyright Copyright 2013 2013 Elsevier; Elsevier; ( (bb)) X-ray X-ray crystalcrystal structure of apo-L161Capo-L161C/L165C-Fr/L165C-Fr withwith CdCdions ions bound bound (PDB (PDB code code 6JEE), 6JEE), and and a close-upa close-up view view of ofthe the fourfold fourfold axis axis channel. channel. Reprinted Reprinted with with permission permission from from Ref. [70Ref.], Copyright[70], Copyright 2019 The 2019 Royal The SocietyRoyal Societyof Chemistry; of Chemistry; (c) X-ray crystal(c) X-ray structure crystal of structure tetrameric of hsALR tetrameric (PDB codehsALR 3R7C), (PDB and code a close-up 3R7C), viewand ofa

close-upthe Cd2Cl view4O6 cluster.of the Cd Reprinted2Cl4O6 cluster. with permission Reprinted from with Ref. permissi [71]. Copyrighton from Ref. 2012, [71]. International Copyright Union2012, Internationalof Crystallography; Union (ofd) Crystallography; X-ray crystal structure (d) X-ray of nvPizza2-S61H58 crystal structure (PDB of nvPizza2-S61H58 code 5CHB), and (PDB a close-up code

5CHB),view of theand Cd a 7close-upCl12 cluster. view Reprinted of the withCd7Cl permission12 cluster. Reprinted from Ref. [72with], Copyright permission 2015 from Wiley-VCH. Ref. [72], 2.3. NobleCopyright Metal 2015 Clusters Wiley-VCH.

2.3. NobleIn general, Metal nobleClusters metals refer to eightelements, including ruthenium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), osmium (Os), iridium (Ir), platinum (Pt), and gold (Au). They tend to form metal In general, noble metals refer to eightelements, including ruthenium (Ru), rhodium (Rh), clusters with a sub-nanometer size (<2 nm), which have a diverse array of applications in biology, palladium (Pd), silver (Ag), osmium (Os), iridium (Ir), platinum (Pt), and gold (Au). They tend to energy, and the environment [73]. Moreover, the formation of noble metal clusters with desirable form metal clusters with a sub-nanometer size (<2 nm), which have a diverse array of applications sizes can be controlled by rationally designed metal-binding proteins. For example, Morozov and in biology, energy, and the environment [73]. Moreover, the formation of noble metal clusters with Ogawa [74] designed helical bundles to form trimers, tetramers, and hexamers, which can bind six, desirable sizes can be controlled by rationally designed metal-binding proteins. For example,

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Morozov and Ogawa [74] designed helical bundles to form trimers, tetramers, and hexamers, which caneight, bind and six, 12 Ageight,+ ions, and respectively. 12 Ag+ ions, Upon respectively. chemical reduction,Upon chemical a series reduction, of Ag0 nanoclusters a series of withAg0 nanoclusterspredictable sizes with can predictable be formed, sizes which can displaybe formed, strong which visible display fluorescence strong visible with a fluorescence number-dependent with a number-dependentemission energy. Recently, emission Cortajarena energy. Recently, et al. developed Cortajarena a simple et al. strategy developed to design a simple proteins strategy for theto designsustainable proteins synthesisof for the metal sustainable nanoclusters synthesisof by the introductionmetal nanoclusters of a dihistidine by the coordination introduction site of [75 a]. dihistidineUsing this approach,coordination they site obtained [75]. Using metal this nanoclusters approach, they such obtained as Cu cluster metal (15 nanoclusters1 atoms), such Ag clusteras Cu ± cluster(nine (151 atoms),± 1 atoms), and Ag Au cluster cluster (nine (five ± 1 atoms),1 atoms) and within Au cluster the protein (five ± sca1 atoms)ffold of within a 34-amino the protein acid ± ± scaffoldhelix–turn–helix of a 34-amino motif, acid which helix–turn–helix showed photostable motif, which luminescence showed photostable both in vitro luminescenceand in vivo, therebyboth in vitroallowing and biomedicalin vivo, thereby applications allowing such biomedical as cell imaging applications and labeling. such as cell imaging and labeling. AsAs shown in thethe previousprevious section, section, Fr Fr is is a highlya highly symmetrical symmetrical cage-like cage-like protein. protein. In itsIn threefoldits threefold axis axischannel, channel, the presencethe presence of both of both His and His Cys and residues Cys resi allowsdues allows the coordination the coordinati of nobleon of metalnoble ions, metal such ions, as suchPd2+ asand Pd Au2+ and3+ ions. Au3+ These ions. metalThese ionsmetal can ions also can be also designed be designed to form to metal form clusters. metal clusters. Ueno et Ueno al. showed et al. II 3 showedthat metal that complexes metal complexes of Pd (allyl) of Pd (allylII(allyl)= η (allyl-C3H =5) η can3-C be3H5 accumulated) can be accumulated in the threefold in the axisthreefold channel axis of channelapo-Fr, formingof apo-Fr, a thiol-bridged forming a dinuclear thiol-bridged structure, dinuclear which wasstructure, stabilized which by the was coordination stabilized of by His114 the coordinationand a water moleculeof His114 (Figure and a 4watera, middle) molecule [ 76]. (Fig Whenure His1144a, middle) was replaced[76]. When with His114 a non-coordinating was replaced withAla residue,a non-coordinating a unique thiolato Ala residue, trinuclear a Pd-clusterunique thiolato was formed, trinuclear with Pd-cluster a six-membered was formed, ring structure with a six-membered(Figure4a, right). ring This structure observation (Figure suggests 4a, right). that theTh threefoldis observation axis channel suggests of apo-Frthat the can threefold be modified axis channelto acquire of metalapo-Fr ions can andbe modified form novel to acquire metal clusters. metal ions and form novel metal clusters. Moreover,Moreover, as as shown in Figure 33b,b, UenoUeno etet al.al. observedobserved the formationformation of a goldgold sub-nanoclustersub-nanocluster atat the the threefold threefold axis axis channel of apo-Fr [77]. [77]. By By the the introduction introduction of of two two surface surface Cys Cys residues, residues, the the apo-E45C/R52C-Frapo-E45C/R52C-Fr protein protein with with Au Au3+3+ ionsions loaded loaded were were first first cross- cross-linkedlinked together together to to form form stable stable crystals,crystals, which which showed showed the the binding binding of of multiple Au Au3+3+ ionsions at at the the threefold threefold axis axis channel channel with with His114, His114, 2+ Cys126,Cys126, and and H H2O2O as as the the ligands, ligands, as as well well as as a aCd Cd2+ ion,ion, due due to tothe the use use of (NH of (NH4)2SO4)24/CdSOSO4/CdSO4 as 4theas 3+ precipitantthe precipitant for crystallization for crystallization (Figure (Figure 4ba′).4 Uponba 0). the Upon reduction the reduction of these of Au these3+ ions Au by usingions different by using concentrationsdifferent concentrations of NaBH4, of the NaBH X-ray4, thestructural X-ray structuralanalysis show analysised the showed gradual the movement gradual movement of the Au of3+ ionsthe Auand3+ ionsthe andultimate the ultimate formation formation of a sub-nano of a sub-nano gold gold cluster cluster (totally (totally 10 10 Au Au3+3 +ions),ions), which which accompaniedaccompanied significant significant conformationalconformational changeschanges of of the the His114 His114 residue residue (Figure (Figure4(bb 4bb0–bd′–d0′).)). Therefore, Therefore, thesethese observationsobservations deepeneddeepened our our understanding understanding of metal of metal cluster cluster formation formation as well asas its well interactions as its interactionswith the protein with microenvironments.the protein microenvironments.

FigureFigure 4. 4. ((aa)) Pd(allyl) Pd(allyl) complexes complexes binding binding to to the the threefold threefold channel channel of of apo-Fr, apo-Fr, which which form form a a trinuclear trinuclear Pd-clusterPd-cluster as as a a result result of of H114A H114A mutation. mutation. Reprinted Reprinted with with permission permission from from Ref. Ref. [76], [76], Copyright Copyright 2008 2008 AmericanAmerican ChemicalChemical Society.Society. (b ()b X-ray) X-ray crystal crystal structures structures Au-loaded Au-loaded apo-Frs apo-Frs in the absencein the absence and presence and

presenceof different of concentrationsdifferent concentrations of NaBH4 ,of showing NaBH4 the, showing process the of formationprocess of of form an Au-clusteration of an at Au-cluster the threefold at thechannel threefold of apo-Fr channel [77 ].of apo-Fr[77].

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Molecules 2019, 24, 2743 6 of 18 2.4. Other Metal Clusters In addition to those metal clusters mentioned in previous sections, other non-nativemetal 2.4. Other Metal Clusters clusters can also be formed in the protein scaffolds with a structural role, which were either rationallyIn addition designed to those or metalfound clusters in experiments. mentioned in For previous example, sections, Sadler other et non-nativemetal al. showed that clusters an ferric–ion-bindingcan also be formed inprotein the protein from scaNeisseriaffolds withgonorrhoeae a structural (nFbp) role, may which form were trinuclear either rationally or pentanuclear designed oxo-clustersor found in experiments.of Hf4+ and Zr For4+ using example, a di-tyrosyl Sadler etcluster al. showed nucleation that anmotif ferric–ion-binding (Tyr195-Tyr196) protein [78,79]. from An 4+ 4+ X-rayNeisseria crystal gonorrhoeae structure(nFbp) of Zr may3–nFbp form was trinuclear shown orin pentanuclearFigure 5a, which oxo-clusters revealed of an Hf oxo-Zrand Zr3 phosphateusing a IV clusterdi-tyrosyl that cluster was nucleationcoordinated motif directly (Tyr195-Tyr196) by both Tyr195 [78,79 ].and An X-rayTyr196 crystal to two structure of the ofthree Zr3 –nFbpZr ions, was whereasshown in the Figure O 5atomsa, which of revealedthe cluster an oxo-Zrwere H-bonded3 phosphate to cluster surrounding that was amino coordinated acids directly[79]. Müller by both et IV Tyr195al.observed and Tyr196several totypes two of thepoly threenucleartungsten Zr ions, whereas oxide clusters the O atoms (W2, ofW3 the, W cluster6, W7, and were W H-bonded7+x) in the bindingto surrounding cavity of amino a Mo/W-storage acids [79]. Müllerprotein, et and al.observed a close-up several view typesof the ofoxo-W polynucleartungsten3-cluster was shown oxide in Figureclusters 5b (W [80].2,W 3,W6,W7, and W7+x) in the binding cavity of a Mo/W-storage protein, and a close-up viewRecently, of the oxo-W Liu 3et-cluster al. found was shownthat ammoniumtetrathiomolybdate in Figure5b [80]. ([(NH4)2MoS4], TM) can induce the dimerizationRecently, Liu of et the al. foundmetal-binding that ammoniumtetrathiomolybdate domain ofcellular copper efflux ([(NH 4protein)2MoS4 ATP7B], TM) can (WLN4), induce due the todimerization the formation of the of metal-bindinga unique sulfur-bridged domain ofcellular Mo2S6O copper2 at the einterface,fflux protein as shown ATP7B by (WLN4), the X-ray due crystal to the structureformation in of Figure a unique 5c sulfur-bridged[81].The detailed Mo structur2S6O2 ate theshowed interface, that asthe shown Mo atom by the formstwo X-ray crystal Mo=O structure double bonds,in Figure as5 cindicated [ 81].The detailedby the structuredistances showedbetween that Mo the and Mo atomO (1.5 formstwo and 1.6 Mo Å),= Owhich double is bonds, in a pyramidalgeometry,as indicated by the distances with coordina betweention Mo from and six O S(1.5 atoms and (four 1.6 Å), Cys which and two is in inorganic a pyramidalgeometry, S atoms) at a withdistance coordination of 2.3 to 2.5 from Å. six S atoms (four Cys and two inorganic S atoms) at a distance of 2.3 to 2.5 Å.

Figure 5. ((aa)) X-ray X-ray structure structure of ZrZr33–nFbp showing the coordinationcoordination geometry.geometry. Reprinted with permission from Ref. Ref. [79]. [79]. Copyright Copyright 2004 2004 Wiley–VCH; Wiley–VCH; (b ()b Structure) Structure of ofthe the Mo/WSto Mo/WSto protein protein of A. of

A.vinelandii vinelandii, and, and a close-up a close-up view view of of the the oxo-W oxo-W3-cluster.3-cluster. Reprinted Reprinted with with permission permission from from Ref. Ref. [80], [80], Copyright 2007 Wiley-VCH; ( c) X-ray structure of a dimer of thethe metal-bindingmetal-binding domain ofcellular

copper eeffluxfflux protein ATP7B (WLN4),(WLN4), andand aa close-upclose-up viewview ofof aa clustercluster MoMo22S66O2 atat the the interface interface [81]. [81].

3. Artificial Artificial MetalloproteinsMetalloproteins/Metalloenzymes/Metalloenzymes with Metal Clusters forfor ElectronTransferElectronTransfer Nature has evolved diverse electron-transfer centers for metalloproteins, which includes single metal ions such as iron and copper, metal complexes such as b-type and c-type hemes, and metal clusters such as different different iron–sulfuriron–sulfur clusters ([2Fe-2S], [3Fe-4S] or [4Fe-4S]), with a broad range of reduction potentialsfrom 700mV to +800 mV [7]. Due to their important rolesof electron transfer in reduction potentialsfrom −700mV to +800 mV [7]. Due to their important rolesof electron transfer in biology, as well as serving as catalysts, iron–sulfur clusters are of considerable interest in the design of artificialartificial metalloproteins and metalloenzymes, which aimed at gaining insights into the complex nature systems,systems, and and possible possible applications applications in industry in industry and energy and [82 energy,83]. With [82,83]. a deep understandingWith a deep understandingof structural principles of structural for natural principles iron–sulfur for natural proteins, iron–sulfur the design proteins, of artificial the iron–sulfurdesign of proteinsartificial iron–sulfurhasobtained proteins significant hasobtaine progressd [significant83]. progress [83]. In a pioneer work, with the help of computer-aided design, Goldren et al. introduced four Cys residues into the hydrophobic core of thioredoxin, followed by the incorporation of a [4Fe-4S]

Molecules 2019, 24, 2743 7 of 18 cluster into the protein scaffold [84]. Similarly, by the introduction of eight Cys residues in the hydrophobic core of a dimeric three-helix bundle (domain-swapped dimer, DSD), Ghirlanda et al. that two cubane-type [4Fe-4S] clusters was readily bound by in situ reconstitution, which increases the thermal stability relative to that of apo-protein. Meanwhile, the two clusters were separated by 29–34 Å, and were not efficientin electron transfer [85]. In a second generation of protein design, these authors shortened the distance between two clusters to 12 Å (Figure6a), which is a distance comparable to native intercluster distance ( 15 Å), such as in ferredoxin [86]. In addition to structural mimicking, ≤ the designed protein, named DSD-Fdm, showed a reduction potential (–479 mV, [4Fe-4S]2+/1+) within the lower range of natural ferredoxins. As a result, the reduced protein was able to transfer electrons to ferric Cytc550 (Figure6a), with a stoichiometry of Cyt c550/DSD-Fdm = 2/1, which closely mimics the function of natural ferredoxins. Note that the protein reduction potential could be fine-tuned by ligand replacement. For example, the replacement of one of the Cys ligands with Ser or Leu increased thereduction potentialto 4 mV and 12 mV, respectively [87]. More recently, Falkowskia et al. designed − artificial symmetric ferredoxins that bind two [4Fe-4S] clusters and exhibit reduction potentials ranging from 405 mV to 515 mV, which are capable of shuttling electrons in vivo, such as through designed − − cellular pathways in E. coli [88]. Similar to other metal clusters mentioned in previous sections, the iron–sulfur cluster can also be designed within the protein interface if it forms a suitable binding pocket. For example, Kerfeld et al. redesigned the interface of a trimeric bacterial microcompartment (BMC) shell protein by introducing a single Cys at position 55, namely BMC-T1-S55C [89]. The designed protein was found to spontaneously incorporate a [4Fe-4S] cluster at the threefold symmetry axisof the trimer when the protein was expressed in vivo. As shown by X-ray structure (Figure6b), the cluster was coordinated by three Cys55 residues, as well as an additional water molecule, which provides the first structural information for a designed [4Fe-4S] cluster within a protein scaffold. Moreover, the cluster exhibited a reduction potential of 370 mV (pH 7.5); this value is close to that of the minimal ferredoxin maquette − ( 350 mV, pH 8) [90], whereas it is more positive than that of dimeric ferredoxin maquette ( 479 mV, − − pH 7.5) [86]. This comparison suggests that greaterexposure of the [4Fe-4S] cluster to the solution leads to more positive potential, which in turn suggests that the reduction potential of the [4Fe-4S] cluster can be fine-tuned by modifying the environment hydrophobicity. Metalloenzymes with multi-cofactors are efficient in catalyzing multi-electron reactions, such as heme-copper oxidases (HCO, CuB-heme dinuclear center) [91–93], nitric oxide reductases (NOR, FeB-heme dinuclear center) [34,94–96], and sulfite reductase (SiR, heme-[4Fe-4S] center) [48,97,98]. It is full of challenges to reproduce both the structure and function of these native enzymes by the design of artificial enzymes with a heteronuclear cofactor. Lu et al. have made significant achievements in this field. In addition to the design of both the structural and functional model of HCO and NOR in the scaffold of Mb, namely CuBMb [92] and FeBMb 34], respectively, they recently designed an artificial SiR in the scaffold of CcO (Figure6c) [ 98]. With the help of the Rosetta matcher, four residues were selected and mutated to Cys (H75C, T180C, W191C, and L232C) to coordinate to the iron of [4Fe-4S], where Cys175 acts as an axial ligand the heme iron. As shown by the modeling structure in Figure6c, the designed enzyme closely mimics the active site of native SiR. To further optimize the microenvironment of the heme active site to improve substrate binding and catalysis, triple mutations (W51K, H52R, and P145K) were performed, which enhances the reductase activity by ~5.3-fold. Moreover, an H-bond was further introduced to the [4Fe-4S] cluster by D235N or D235C mutation, resulting in a 17-fold and 63-fold increase over that of the first generation, which indicates that both the microenvironment and H-bond interactions are key factors affecting the catalytic efficiency. Note that the highest rate of 1 1 sulfite reduction achieved was 21.8 min− , which is ~18% that of a native SiR (121 min− ). This study demonstrates that the rational design of a [4Fe-4S] cluster in combination with secondary interactions may confer upon artificial enzymesan ability to performmulti-electron and multi-proton reduction. Molecules 2019, 24, 2743 8 of 18 Molecules 2019, 24, x FOR PEER REVIEW 8 of 18

FigureFigure 6.6.( a(a)) Rational Rational design design of twoof two [4Fe-4S] [4Fe-4S] clusters clusters in domain-swapped in domain-swapped dimer(DSD)-Fdm dimer(DSD)-Fdm that transfer that electronstransfer electrons to ferric Cytto ferricc. Reprinted Cyt c. Reprinted with permission with permission from [86]. fr Copyrightom [86]. Copyright 2014 American 2014 ChemicalAmerican Society;Chemical (b )X-raySociety; structure (b)X-ray of structure BMC-T1-S55C of BMC-T1-S55C and a close-up and view a ofclose-up the [4Fe-4S] view clusterof the (PDB[4Fe-4S] code cluster 5DII). Reprinted(PDB code with 5DII). permission Reprinted from with [89 permission]. Copyright from 2014 [89]. American Copyright Chemical 2014 Americ Society;an ( cChemical) Rational Society; design of(c) a Rational [4Fe-4S] clusterdesign inof cytochrome a [4Fe-4S] cclusterperoxidase in cytochrome (CcP) that closelyc peroxidase mimics (C thecP) active that siteclosely of native mimics sulfite the reductaseactive site (SiR). of native Reprinted sulfite with reductase permission (SiR). from Reprinted [98]. Copyright with permission 2018 AAAS. from [98]. Copyright 2018 AAAS. 4. Artificial Metalloenzymes with Metal Clusters for Catalysis

4.1.4. Artificial Iron–Sulfur Metalloenzymes Clusters with Metal Clusters for Catalysis

4.1. Iron–SulfurAs mentioned Clusters in the previous section, iron–sulfur clusters play rolesof both electron transfer and catalysis in biology. For example, nitrogenase catalyzes the reduction of nitrogento ammonia, As mentioned in the previous section, iron–sulfur clusters play rolesof both electron transfer as well as other substrates (C H , CN , etc.), at its cofactor center of [(Cit)MoFe S C]n (designated and catalysis in biology. For 2example,2 − nitrogenase catalyzes the reduction of nitrogento7 9 − ammonia, M-cluster), where electrons were transferred from a [4Fe–4S] cluster via an [8Fe–7S] cluster (designated as well as other substrates (C2H2, CN−, etc.), at its cofactor center of [(Cit)MoFe7S9C]n− (designated P-cluster) [99]. The M-cluster consists of [Mo–3Fe–3S] and [4Fe–3S] subclusters, as bridged by three S M-cluster), where electrons4 were transferred from a [4Fe–4S] cluster via an [8Fe–7S] cluster atoms and one carbide (C −) atom (Figure7a) [ 100]. To mimic this unique geometry and the anionic (designated P-cluster) [99]. The M-cluster consists of [Mo–3Fe–3S] and [4Fe–3S] subclusters,4 as nature, Holm et al. synthesized a hexanulcear, dithiolateiron–sulfur cluster, [Fe6S9(SEt)2] −, which bridged by three S atoms and one carbide (C4−) atom (Figure 7a) [100]. To mimic this unique was designated Fe RHH (Figure7)[ 101]. To reveal whether the M-cluster mimic could be combined geometry and the6 anionic nature, Holm et al. synthesized a hexanulcear, dithiolateiron–sulfur with a protein scaffold to afford a functional enzyme, Hu et al. removed the native M-cluster from cluster, [Fe6S9(SEt)2]4−, which was designated Fe6RHH (Figure 7) [101]. To reveal whether the M-cluster the catalytic component of nitrogenase, followed by incorporation of the Fe RHH cluster into the mimic could be combined with a protein scaffold to afford a functional enzyme,6 Hu et al. removed apo-protein (Figure7a) [ 102]. The reconstituted artificial enzyme was able to catalyze the reduction of the native M-cluster from the catalytic component of nitrogenase, followed by incorporation of the C2H2 to C2H4 in the presence of the reductase component of nitrogenase and ATP, with an activity Fe6RHH cluster into the apo-protein (Figure 7a) [102]. The reconstituted artificial enzyme was able to comparable to that of the native enzyme. Moreover, in the absence of ATP and presence of a strong catalyze the reduction of C2H2 to C2H4 in the presence of the reductase component of nitrogenase reductant, europium(II) diethylenetriaminepentaacetic acid (EuII-DTPA), the artificial enzyme can and ATP, with an activity comparable to that of the native enzyme. Moreover, in the absence of ATP catalyze the reduction of CN− to C1-C3 hydrocarbons, with CH4 being the major product. This study and presence of a strong reductant, europium(II) diethylenetriaminepentaacetic acid (EuII-DTPA), demonstrates that the functional artificial enzyme can be obtained by the combination of a synthetic the artificial enzyme can catalyze the reduction of CN− to C1-C3 hydrocarbons, with CH4 being the cluster with an appropriate protein scaffold. major product. This study demonstrates that the functional artificial enzyme can be obtained by the Recently, thesemi-synthetic approach was extended to reveal the assembly mechanism of the combination of a synthetic cluster with an appropriate protein scaffold. M-cluster. For determination of the ninth sulfur source of the l-cluster [Fe8S9C], Hu et al. synthesized Recently, thesemi-synthetic approach was extended to reveal the assembly mechanism of synthe a water-stable and soluble [4Fe-4S] cluster with four –SCH2CH2OH ligands (designated [4Fe-4S] , M-cluster. For determination of the ninth sulfur source of the L-cluster [Fe8S9C], Hu et al. Figure7b, left) that were exchangeable by Cys residues from protein, and incorporated it into the synthesized a water-stable and soluble [4Fe-4S] cluster with four2 –SCH2CH2OH2 ligands2 (designated apo-protein of nitrogenase [103]. The results showed that SO −, instead of S − or SO −, gives rise [4Fe-4S]syn, Figure 7b, left) that were exchangeable by Cys residues3 from protein, and4 incorporated to the ninth sulfur, with the formation of a new [Fe8S8C] cluster intermediate, namely the L*-cluster. it into the apo-protein of nitrogenase [103]. The results showed that SO32−, instead of S2− or SO42−, Then, the M-cluster was maturated by the insertion of Mo and homocitrate (HC) (Figure7c). Moreover, gives rise to the ninth sulfur, with the formation of a new [Fe8S8C] cluster intermediate, namely the the cluster of [4Fe-4S]syn was found to be able to catalyze the reduction of C1 substrates such as CO L*-cluster. Then, the M-cluster was maturated by the insertion of Mo and homocitrate (HC) (Figure2 7c). Moreover, the cluster of [4Fe-4S]syn was found to be able to catalyze the reduction of C1 substrates such as CO2 and CO in the presence of EuII-DTPA, producing hydrocarbons (CH4, C2H4,

Molecules 2019, 24, 2743 9 of 18 Molecules 2019, 24, x FOR PEER REVIEW 9 of 18

II andand COC2H in6) thein an presence aqueous of buffer Eu -DTPA, (Figure producing 7b, right) hydrocarbons [104,105]. These (CH 4observations,C2H4, and Cindicate2H6) in anan aqueousinherent bucatalyticffer (Figure feature7b, for right) the[ [4Fe-4S]104,105]. cluster, These observationswhich may thus indicate be used an inherentfor the design catalytic of featureother artificial for the [4Fe-4S]metalloenzymes. cluster, which may thus be used for the design of other artificial metalloenzymes.

FigureFigure 7.7.( a()a An) An artificial artificial metalloenzyme metalloenzyme constructed constructed by replacement by replacement of the M-cluster of the withM-cluster a synthesized with a 4 [Fesynthesized6S9(SEt)2] [Fe− cluster6S9(SEt) in2]4− nitrogenase cluster in nitrogenase scaffold. Reprinted scaffold. withRepr permissioninted with frompermission [102]. from Copyright [102]. 2 2015Copyright Wiley-VCH; 2015 Wiley-VCH; (b) The structure (b) The of [4Fe-4S(SCHstructure of 2[4Fe-4S(SCHCH2OH)4] −2CHcluster2OH) (left),4]2− cluster and its(left), catalysis and ofits II COcatalysis2/CO reductionof CO2/CO in reduction the presence in the ofeuropium(II) presence of europium diethylenetriaminepentaacetic(II) diethylenetriaminepentaacetic acid (Eu -DTPA) acid (right)(EuII-DTPA) [104]. (right) Copyright [104]. 2018 Copyright Nature 2018 press; Nature (c) The press; proposed (c) The mechanism proposed of mechanisml-cluster assembly of L-cluster in nitrogenase,assembly in followednitrogenase, by followed the maturation by the ofmaturation M-cluster. of SAM:M-cluster. S-adenosyl- SAM: S-adenosyl-l-methionine;L-methionine; NifH: the reductaseNifH: the reductase component component of Mo-nitrogenase;HC:homocitrate. of Mo-nitrogenase;HC:homocitrate. Reprinted Reprinted with permission with permission from [from103]. Copyright[103]. Copyright 2018 Macmillan 2018 Macmillan Publishers. Publishers. 4.2. Copper–Sulfur Clusters 4.2. Copper–Sulfur Clusters Copper is an essential element in biological systems. When associated with a protein matrix Copper is an essential element in biological systems. When associated with a protein matrix by by coordination with His, Cys, and/or Met, it plays diverse functions including O2 binding, electron coordination with His, Cys, and/or Met, it plays diverse functions including O2 binding, electron transfer, and catalysis [106,107]. Copper centers have various types, such as the CuA and CuB transfer, and catalysis [106,107]. Copper centers have various types, such as the CuA and CuB centers found in cytochrome c oxidase (CcO) that contain two and one copper atom, performing centers found in cytochrome c oxidase (CcO) that contain two and one copper atom, performing electron-transfer and catalysis functions, respectively [106,107]. It was revealed that the axial Met electron-transfer and catalysis functions, respectively [106,107]. It was revealed that the axial Met ligand and the surrounding amino acids influence the reduction potential of CuA via coordination, ligand and the surrounding amino acids influence the reduction potential of CuA via coordination, H-bond, and hydrophobic interactions [107–109], and in the active site of the CuB center, a novel H-bond, and hydrophobic interactions [107–109], and in the active site of the CuB center, a novel Tyr–His cross-link was found to fine-tune the protein reactivity [110,111]. In addition, the CuZ center Tyr–His cross-link was found to fine-tune the protein reactivity [110,111]. In addition, the CuZ was discovered in N2O reductase (N2OR), which containsfour copper atoms forming a distorted center was discovered in N2O reductase (N2OR), 2-which containsfour copper atoms forming a tetrahedral geometry, with an inorganic sulfur ion (S ) as a bridging ligand (Cu4S cluster, Figure8a, distorted tetrahedral geometry, with an inorganic sulfur ion (S2-) as a bridging ligand (Cu4S cluster, PDB code 1QNI [112]). The copper ions (CuI-IV) were coordinated by seven His residues, and the Figure 8a, PDB code 1QNI [112]). The copper ions (CuI-IV) were coordinated by seven His residues, CuI–CuIV edge was bridged by one unknown O ligand (Figure8a,b), which was proposed to be and the CuI–CuIV edge was bridged by one unknown O ligand (Figure 8a,b), which was proposed+ to the active site where the substrate binds and reduces to N2 and H2O (N2O + 2e− + 2H N2 + be the active site where the substrate binds and reduces to N2 and H2O (N2O + 2e− + 2H+ → N→2 + H2O) H2O) [113,114]. [113,114]. It is full of challenges to design an artificial enzyme with a Cuz center, and no report is availablein It is full of challenges to design an artificial enzyme with a Cuz center, and no report is the literature yet. Meanwhile, some progresses have been made in the design of synthetic model availablein the literature yet. Meanwhile, some progresses have been made in the design of complexes mimicking the structure and function of the Cuz cluster in native N2OR [115,116]. For synthetic model complexes mimicking the structure and function of the Cuz cluster in native N2OR example, Cramer et al. reported the first functional model of Cuz with a [Cu3(µ3-S2)] core (Figure8c), [115,116]. For example, Cramer et al. reported the first functional model of Cuz with a [Cu3(μ3-S2)] which exhibited spectroscopic features similar to those of N2OR, and can reduce N2O to N2 under mild core (Figure 8c), which exhibited spectroscopic features similar to those of 2N2OR, and can reduce conditions such as at room temperature [117]. Instead of using a bridging S − ligand, Esmieu et al. N2O to N2 under mild conditions such as at room temperature [117]. Instead of using a bridging S2− synthesized a dissymmetric mixed-valentdicopper(II,I) complex using thiolates, which contains a ligand, Esmieu et al. synthesized a dissymmetric mixed-valentdicopper(II,I) complex using thiolates, [Cu2S] core with labile triflate and water molecules at the copper centers (Figure8d) [ 118]. This simple which contains a [Cu2S] core with labile triflate and water molecules at the copper centers (Figure 8d) [118]. This simple dicopper complex was shown to be a functional model of the Cuz cluster, by the

Molecules 2019, 24, 2743 10 of 18 Molecules 2019, 24, x FOR PEER REVIEW 10 of 18 reductiondicopper complexof N2O to was N2at shown room totemperature. be a functional Recently, model Mankad of the Cuz et al. cluster, reported by thea tetranuclear reduction ofcopper N2O clusterto N2at with room a temperature.[Cu4(μ4-S)] core Recently, (Figure Mankad 8e), in its et al.one-hole reported (S=1/2) a tetranuclear redox state copper [119]. clusterThe one-hole with a clusterwas[Cu4(µ4-S)] shown core (Figure to reduce8e), in N its2O one-hole and produce (S =1 /N2)2 redox, which state is the [119 first]. The model one-hole closely clusterwas mimicking shown the one-electronto reduce N2 Oreduced and produce form of N2 native, which Cuz is the (termed first model Cuz*, closely CuI4S). mimicking Therefore, the although one-electron these reduced model I complexesform of native work Cuz in (termed organic Cuz*,solvents Cu 4suchS). Therefore, as CH2Cl although2 and acetone, these modelthey are complexes very useful work for in gaining organic thesolvents structure such and as CHfunction2Cl2 and relationship acetone, of they native are veryN2OR. useful Moreover, for gaining the incorporation the structure of and these function model complexesrelationship in of suitable native N protein2OR. Moreover, scaffolds themight incorporation be able to ofproduce these model artificial complexes metalloenzymes in suitable working protein insca physiologicalffolds might beconditions. able to produce artificial metalloenzymes working in physiological conditions.

Figure 8. ((aa)) The The X-ray X-ray structure structure of of the the Cuz Cuz center center in in N N22OROR from from P.P. nautica (PDB(PDB code code 1QNI 1QNI [112]); [112]); (b) TheThe coordinationcoordination geometry geometry of theof the Cuz Cuz center center in native in native N2OR; N (c2–OR;e) The (c– structurese) The structures of the synthetic of the syntheticfunctional functional models of models the native of the Cuz native center Cuz [117 center–119]. [117–119]. 4.3. Other Metal Clusters 4.3. Other Metal Clusters The oxygen-evolving center (OEC) in photosystem II has received much attention in the last The oxygen-evolving center (OEC) in photosystem II has received much attention in the last decade, which contains a Mn4CaO5 cluster for water oxidation (Figure9a). Many e ffects have been decade, which contains a Mn4CaO5 cluster for water oxidation (Figure 9a). Many effects have been directed to synthetic mimics of the OEC. For example, Agapie et al. have rationally synthesized a directed to 6synthetic+ mimics of the OEC. For example, Agapie et al. have rationally synthesized a [Mn3CaO4] cubane that structurally models the Mn3Ca subsite of OEC [120]. Zhang et al. have [Mn3CaO4]6+ cubane that structurally models the Mn3Ca subsite of OEC [120]. Zhang et al. have synthesized a Mn4CaO4 cluster closely mimicking the native OEC, not only in the metal–oxygen synthesized a Mn4CaO4 cluster closely mimicking the native OEC, not only in the metal–oxygen core, but also in the coordinating amino acids with ligands of acetate and pyridine (Figure9b) [ 121]. core, but also in the coordinating amino acids with ligands of acetate and pyridine (Figure 9b) [121]. Note that the two water ligands on the Ca2+ ion could be mimicked not only by acetate, but also by Note that the two water ligands on the Ca2+ ion could be mimicked not only by acetate, but also by polar solvents such as acetonitrile and DMF, as structurally characterized recently by Zhang et al. [122]. polar solvents such as acetonitrile and DMF, as structurally characterized recently by Zhang et al. Moreover, the artificial Mn4CaO4 cluster underwent four redox transitions similar to that of native [122]. Moreover, the artificial Mn4CaO4 cluster underwent four redox transitions similar to that of OEC, which thus provides an ideal model for studying the structure and function relationship of native native OEC, which thus provides an ideal model for studying the structure and function OEC, as well as an artificial enzyme model for applications in water oxidation. relationship of native OEC, as well as an artificial enzyme model for applications in water Since the above-synthesized cluster was not incorporated into a protein scaffold, the secondary oxidation. coordination sphere interactions could hardly be fine-tuned. Meanwhile, in a native photosystem Since the above-synthesized cluster was not incorporated into a protein scaffold, the secondary II system, Tyr residue facilitates protein-coupled electron transfer (PCET) between the OEC center coordination sphere interactions could hardly be fine-tuned. Meanwhile, in a native photosystem II and light-harvesting chlorophylls (P680). It is challengeable to design an artificial metalloenzyme system, Tyr residue facilitates protein-coupled electron transfer (PCET) between the OEC center with tunable PCET properties. Recently, Tilley et al. synthesized a biotinylated Co O cluster and light-harvesting chlorophylls (P680). It is challengeable to design an artificial metalloenzyme4 4 (Figure9c) and incorporated it into a Sav protein by employing the biotin-streptavidin technology [ 123]. with tunable PCET properties. Recently, Tilley et al. synthesized a biotinylated Co4O4 cluster By introduction of a proximal Tyr (S112Y mutation), the X-ray crystal structure revealed that Tyr112 (Figure 9c) and incorporated it into a Sav protein by employing the biotin-streptavidin technology forms an H-bond interaction with the water molecule that acts as an axial ligand of the Co ion [123]. By introduction of a proximal Tyr (S112Y mutation), the X-ray crystal structure revealed that Tyr112 forms an H-bond interaction with the water molecule that acts as an axial ligand of the Co ion (Figure 9d). As a result, a chemistry of multi-e−/multi-H+ was observed in electronchemical

Molecules 2019, 24, 2743 11 of 18

Molecules 2019, 24, x FOR PEER REVIEW 11 of 18 + (Figure9d). As a result, a chemistry of multi-e −/multi-H was observed in electronchemical studies, withstudies, a transition with a transition of the mechanism of the mechanism at pH 9.5 correspondingat pH 9.5 corresponding to the pKa valueto the (~9.5) pKa value of Tyr (~9.5) (Figure of9 e,f).Tyr + Note(Figure that 9e,f). in the Note absence that of in the the proximal absence Tyr of (Ser112 the proximal or Phe112), Tyr only (Ser112 1e−/1H or chemistryPhe112), wasonly observed, 1e−/1H+ whichchemistry suggests was observed, that the secondary which suggests sphere that interactions, the secondary as provided sphere interactio by the redoxns, as active provided Tyr residue, by the + areredox critical active for Tyr fine-tuning residue, theare multi-ecritical− for/multi-H fine-tuningreactivity the multi-e for the−/multi-H artificial enzyme+ reactivity [123 for]. Notethe artificial that the introductionenzyme [123]. ofa Note redox that active the introduction residue such ofa as Tyrredo andx active Trp inresidue the heme such distal as Tyr pocketor and Trp on in the the protein heme surfacedistal pocketor was also on shown the protein to fine-tune surface the reactivitywas also ofshown artificial to fine-tune peroxidases the designed reactivity in of the artificial protein scaperoxidasesffold of myoglobin designed in [46 the,124 protein,125]. scaffold of myoglobin [46,124,125].

Figure 9. a Figure 9. ((a)) The X-ray crystal structure of native oxygen-evolving center (OEC) showing the core Mn4CaO5 cluster and the ligands; (b) The X-ray crystal structure of a synthesized Mn4CaO4 cluster; Mn4CaO5 cluster and the ligands; (b) The X-ray crystal structure of a synthesized Mn4CaO4 cluster; Reprinted with permission from [121], Copyright 2015 AAAS; (c) The chemical structure of biotinylated Reprinted with permission from [121], Copyright 2015 AAAS; (c) The chemical structure of Co4O4 cluster, biot-β-Ala-1; (d) The X-ray crystal structure of 2xm-S112Y-Sav with biot-β-Ala-1 bound biotinylated Co4O4 cluster, biot-β-Ala-1; (d) The X-ray crystal structure of 2xm-S112Y-Sav with (PDB code 6AUE); (e–f) Proposed mechanisms for the multi-e /multi-H+ reactivity of the designed biot-β-Ala-1 bound (PDB code 6AUE); (e–f) Proposed mechanisms− for the multi-e−/multi-H+ enzyme at pH <9.5 or pH >9.5.Reprinted with permission from [123]. Copyright 2018 American reactivity of the designed enzyme at pH <9.5 or pH >9.5.Reprinted with permission from [123]. Chemical Society. Copyright 2018 American Chemical Society. 5. Conclusions and Perspectives 5. Conclusions and Perspectives In summary, artificial metalloproteins and metalloenzymes with diverse metalclusters have been rationallyIn summary, designed artificial in recent metallopr years, inoteins which and the metalloenzymes clustersare formed with either diverse in themetalclusters protein pocket, have betweenbeen rationally the interface designed of protein in recent dimers, years, trimers, in which and the oligomers, clustersare or withinformed the either scaff oldin the of deprotein novo designedpocket, between proteins. the As interface shown of in Figureprotein1 ,dimers, the metal trimer ionss, used and foroligomers, the design or within of metalclusters the scaffold are of not de onlynovo thosedesigned in natural proteins. biological As shown systems, in Figure such 1, as the the metal first-row ions transition used for metalsthe design (Mn, of Co, metalclusters Fe, Cu, and Zn,are not Figures only2 those and6 ,in Figures natural7– biological9), but also systems, non-native such metalas the ions, first-row such transition as those ofmetals the second-row (Mn, Co, Fe, transitionCu, and Zn, metals figures (Zr, Mo,2, 6–9), Pd, Ag,but and also Cd, non-native Figures3,4 ametal and5 a,c)ions, and such the as third-row those of transition the second-row metals (Hf,transition W, and metals Au, Figures (Zr, Mo,4b andPd,5 Ag,b). Althoughand Cd, figures only several 3, 4a,amino 5a, and acids 5c) (Cysand the/Met, third-row His, and transition Asp/Glu) actmetals as the(Hf, ligand W, and for Au, the figures metal clusters,4b and 5b). the Although bridging atomsonly several/ligands amino are diverse, acids (Cys/Met, which are His, usually and Asp/Glu) act as the ligand2 for the2 metal clusters,2 the bridging atoms/ligands are diverse, which are inorganic anions such as S −, SO4 −, Cl−,O −, and OH−, and amino acids (Cys/Asp/Glu) (Figures2–9), usually inorganic anions such as S2−, SO42−, Cl−, O2−, and OH−, and amino acids (Cys/Asp/Glu) as well as synthetic ligands such as –SCH2CH2OH (Figure7b). In most cases, these metal clusters were found(figures to 2–9), play aas structural well as sy role,nthetic which ligands stabilize such the as protein –SCH 3D2CH structures,2OH (Figure dimers, 7b). trimers,In most orcases, oligomers. these Moreover,metal clusters the metalwere found clusters, to play especially a structural iron–sulfur role, which clusters, stabilize have been the protein successfully 3D structures, designed dimers, to play rolestrimers, of bothor oligomers. electron-transfer Moreover, and catalysisthe metal (Figures clusters,6 and especially7). Within iron–sulfur this progress, clusters, some functionalhave been modelsuccessfully complexes designed have beento play rationally roles of designed both electr andon-transfer synthesized, and which catalysis closely (figures6,7). mimic the activeWithin metal this progress, some functional model complexes have been rationally designed and synthesized, which clusters in more complex native metalloenzymes, such as the Cuz center in N2OR (Figure8b–e) and closely mimic the active metal clusters in more complex native metalloenzymes, such as the Cuz Mn4CaO5 cluster in photosystem II (Figure9a,b), respectively. center in N2OR (Figure 8b–e) and Mn4CaO5 cluster in photosystem II (Figure 9a,b), respectively. Currently, the design of artificial metalloproteins and metalloenzymes with metal clusters is still at the stage of providing insights into the structure and functional relationship for native metalloenzymes, and some synthetic models are still waiting for incorporation into suitable protein

Molecules 2019, 24, 2743 12 of 18

Currently, the design of artificial metalloproteins and metalloenzymes with metal clusters is still at the stage of providing insights into the structure and functional relationship for native metalloenzymes, and some synthetic models are still waiting for incorporation into suitable protein scaffolds to work in physiological conditions. Comparatively, artificial metalloenzymes with a single active site have been designed to exhibit catalytic parameters similar to those of native enzymes [50,98,126,127], or even with a much higher catalytic efficiency [128], which have potential applications in the future. Moreover, some artificial metalloenzymes may catalyze reactions beyond the functionalities of natural enzymes, such as the catalytic formation of a C–Si bond by an engineered Cyt c [129]. By the construction of multi-metal clusters in protein scaffolds, the catalysis of multi-electron/multi-proton reactions has been achieved [98,123], which otherwise can hardly be achieved by using a single metal center, suggesting a more promising application in biotransformations. As suggested by Martinez et al., oxidoreductases are on their way from laboratory to industry [130]. We are confident that in the near future, more advanced artificial metalloenzymes with metal clusters will be rationally designed andexplored for practical applications in different fields, such as in biological medicine, biofuel generation, and environmental protection.

Funding: The work on the heme protein design from my group was supported by the National Natural Science Foundation of China (31370812, 21101091) and the double first-class construct program of the University of South China. Acknowledgments: I thank all the co-workers for their studies described in this review. Conflicts of Interest: The authors declare no conflict of interest.

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