Biosynthetic Inorganic Chemistry

Chemical BiologyDOI: 10.1002/anie.200600168

Biosynthetic Inorganic ChemistryYi Lu*

AngewandteChemie

Keywords:biocatalysis · bioinorganic chemistry ·biomimetic synthesis · proteindesign

Y. LuReviews

5588 www.angewandte.org � 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2006, 45, 5588 – 5601

http://www.angewandte.org

1. Introduction

Metal ions play important roles in catalyzing numerouschemical and biological reactions, as the reactivity anddiversity can be modulated at high levels by not only differentmetal ions, but also the different oxidation states of thesesame metal ions.[1–6] The successful synthesis and applicationof novel metal complexes or metalloenzymes can have a greatimpact on all areas of chemistry and biology, whether it isorganic, medicinal, materials chemistry, or biochemistry andcell biology. An important example of such an impact is thedevelopment of metal catalysts for reactions such as nitrogenfixation, methane hydroxylation, and CO oxidation orinsertion (Table 1).[7, 8] These catalysts are key componentsfor multibillion-dollar chemical industries that produce vitalraw chemicals, such as fertilizers.

Interestingly, many of the reactions in Table 1 can becatalyzed by either synthetic catalysts or by biologicalenzymes. A closer look at the two systems suggests that thecatalysts mediate the same reaction under different condi-tions: chemical systems generally operate at high temper-atures and pressures in organic solvent, whereas biologicalsystems work at ambient temperatures and pressures in water.Furthermore, biocatalysts often have high catalytic turnovers.Therefore, the question is not whether one can developcatalysts that can work under milder conditions with highturnovers, but rather how such catalysts can be developed.

A cursory look at the examples in Table 1 may lead one toconclude that one system may be better than the other;however, each system has its own advantages and disadvan-tages (Table 2). Chemical catalysts are generally smaller,easier to synthesize and characterize, cheaper to produce, andmore resistant to harsh conditions such as high temperaturesand pressures. These practical attributes make it difficult toreplace catalysts with enzymes in industrial-scale synthesis,although enzymes possess a number of desirable features forcatalysts. First, they form more-rigid active sites to stabilizethe transition state and thus are more able to acceleratecatalysis at ambient temperature and pressure. Second, they

direct reactions towards a specificfunctional group of an organic sub-strate, thus eliminating or minimizingthe need for protecting groups, which

are often required in synthetic chemistry. Third, the catalyticsite in enzymes is protected, which results in higher catalyststability and higher turnover numbers. Fourth, enzymesachieve high regio-, stereo-, and enantioselectivity. Fifth, thecomplex protein framework is amenable to site-specific (asopposed to solvent-induced) modulation of the secondarycoordination sphere of a metal-binding site. For example, thepKa values for glutamic acid in the same protein vary between4.0 and 8.2, depending on the local electrostatic environmentand hydrophobicity.[9] Finally, enzymes are environmentallybenign, as they are synthesized and applied under physio-logical conditions in aqueous solvents and with biocompatibleand biodegradable ligands.

Since each of the chemical and biological systems has itsown advantages and disadvantages, one may wonder if theadvantages of both systems can be combined. Towards thisend, a number of synthetic biomimetic model systems formetalloproteins have been prepared from small organicmolecules and characterized.[10] Such a synthetic approachhas been very effective in the elucidation of structural andfunctional properties of metalloproteins.

Despite tremendous progress in biomimetic modeling, it isstill relatively difficult to mimic some of the features ofmetalloproteins, for example, site-specific modulation of thesecondary coordination sphere and the regio-, stereo-, andenantioselectivity of a system. Models that reproduce boththe structure and the function of metalloproteins are rare.These challenges, like perhaps all obstacles in syntheticinorganic chemistry, can be overcome by ligand design.Prudent choice of ligand has repeatedly resulted in bettermodels of metalloproteins,[10] especially when features in thesecondary coordination sphere such as hydrogen-bonding

[*] Prof. Y. LuDepartment of ChemistryUniversity of Illinois at Urbana-ChampaignUrbana, IL 61801 (USA)Fax: (+1)217-244-3186E-mail: [email protected]

Inorganic chemistry and biology can benefit greatly from each other.Although synthetic and physical inorganic chemistry have been greatlysuccessful in clarifying the role of metal ions in biological systems, thetime may now be right to utilize biological systems to advance coor-dination chemistry. One such example is the use of small, stable, easy-to-make, and well-characterized proteins as ligands to synthesize novelinorganic compounds. This biosynthetic inorganic chemistry ispossible thanks to a number of developments in biology. This reviewsummarizes the progress in the synthesis of close models of complexmetalloproteins, followed by a description of recent advances in usingthe approach for making novel compounds that are unprecedented ineither inorganic chemistry or biology. The focus is mainly on synthetic“tricks” learned from biology, as well as novel structures and insightsobtained. The advantages and disadvantages of this biosyntheticapproach are discussed.

From the Contents

1. Introduction 5589

2. Application of BiosyntheticInorganic Chemistry inBiomimetic Model Systems 5591

3. Biosynthetic InorganicChemistry for the Synthesis ofNew Inorganic Complexes 5595

4. Conclusions and Outlook 5598

Biomimetic SynthesisAngewandte

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5589Angew. Chem. Int. Ed. 2006, 45, 5588 – 5601 � 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

were introduced.[11,12] The main difference between syntheticmodels and metalloproteins is that the synthetic models oftencontain water-insoluble organic ligands, whereas metallopro-teins consist of polypeptides or proteins. Polypeptides andproteins are unusual organic molecules in that they arenaturally soluble in water and can be synthesized under mildconditions. Through specific folding of the polypeptides andproteins, a rigid network can form that allows site-specificmodulation of the secondary coordination sphere. Thissuggests that polypeptides or proteins were established inthe course of evolution as ligands for metal-based catalysts orenzymes.

So why then should the ligands from biological systemsnot be used to synthesize model compounds? It used to bevery difficult and expensive to make proteins and their

variants, which rendered the use of such proteins as ligandsimpractical. However, advancements in biology, such as thedevelopment of cloning, protein expression, site-directedmutagenesis, and polymerase chain reaction, have made itpossible to reduce dramatically the time required to synthe-size proteins and change amino acid residues. Whereas it usedto take many months or even many years to construct,express, and purify a series of proteins, it now takes a matterof weeks or even days; such progress is similar to that made inthe computer industry (Figure 1). Furthermore, hundreds ofmilligrams or even grams of pure protein can now be

Yi Lu received his BS degree from BeijingUniversity (P.R. China) in 1986 and his PhDfrom the University of California at LosAngeles in 1992 under the guidance of Prof.Joan Selverstone Valentine. After two yearsof postdoctoral research at the CaliforniaInstitute of Technology in the group of Prof.Harry B. Gray, he began his independentcareer as an Assistant Professor in theDepartment of Chemistry at the Universityof Illinois at Urbana–Champaign, where heis now an Alumni Research Scholar Professorof Chemistry. His interests lie in bioinorganicchemistry.

Table 1: Comparison of chemical and biological systems.

Reaction Chemical System Biological System

nitrogen fixation N2(g) + 3H2(g)!2NH3(g)

(a-Fe catalyst, 400–5508C, 100 atm)N2 + 8H+ + 8e� + 16MgATP!2NH3 + H2 +16MgADP + 16 Pi

(nitrogenase)methane hydroxylation CH4 + H2O!CO + 3H2

(Ni catalyst, 700–9008C, 1–25 bar)CO + 2H2!CH3OH(Cu/Zn Catalyst, 250–2808C, 70–110 bar)

CH4 + O2 + NADH + H+!CH3OH + H2O + NAD+

(methane monooxygenase)

CO oxidation CO + H2O!CO2 + H2

(Fe/Cu catalyst, >200 8C)CO + H2O!CO2 + 2H+ + 2e�

(CO dehydrogenase)CO insertion CH3OH + CO!CH3COOH

(Rh(I)I2(CO)2]� , 120 8C, 30 atm)

CH3�[M] + CO + HS�CoA!CH3(CO)�S�CoA + H+ + [M�][a]

(acetyl-CoA synthase)

[a] CH3�[M] is a corrinoid–iron–sulfur protein that acts in the reaction as a methyl group donor; HS-CoA is coenzyme A.

Table 2: Advantages of chemical and biological systems.

ChemistryCatalysts/Materials

BiologyEnzymes/Biomaterials

easy to synthesize mild conditionseasy to characterize rigidity/protectionmore robust site-specific modulation of the

2nd coordination spherecheaper to produce regio- and enantioselectivity

environmentally friendlyapproach: create new structures andfunctions by using a minimalistapproach

approach: study native pro-teins and their variants

Figure 1. Comparison of progresses made in the computer industryand biology. A) According to Moore’s law, the number of transistorsand thus the speed of a computer doubles approximately every18 months. B) Estimated average speeds of cloning, expression, andpurification of recombinant proteins (calculated as the inverse ofnumber of days it takes to complete the process). Techniques such asrecombinant DNA (Rec. DNA), site-directed mutagenesis (SDM), andthe polymerase chain reaction (PCR) have significantly accelerated thebiological progress.

Y. LuReviews



prepared. Thanks to the progress in biotechnology, certainchemical synthesis steps in the production of pharmaceuticalproducts can be replaced by biosynthesis. As a result, thenumber of synthesis steps is reduced, yields are increased, andless chemical waste is produced,[13, 14] so that the difference inefficiency between the synthesis of small organic moleculesand proteins is decreased. At the same time, progress inbiophysics has made possible the routine characterization ofmetalloproteins by different spectroscopic techniques.[15–17]

Advances in structural biology, such as the availability ofhigh-energy synchrotron sources and high-field magnets andtheir associated methodology developments, have also madeit possible to obtain three-dimensional crystal or NMRstructures of proteins in a short period of time. Thus, it isnow possible to synthesize and characterize novel inorganiccompounds using small, stable, easy-to-produce proteins withcharacteristic scaffolds as “ligands”.

The biosynthetic approach uses the same techniques asthe biochemical studies of native enzymes and their mutantforms. However, the following examples below show thatthere are distinct differences between the two approaches:First, like all biomimetic approaches, the biosyntheticapproach is a minimalist approach; it focuses on determiningwhether the necessary structural features identified in nativeenzymes are sufficient to confer the structure and function ofthe enzyme. Therefore, the two approaches are complemen-tary. Second, stable, easy-to-produce, and well-characterizedproteins are used in the biosynthetic approach, and successfulmodels are often as good as, if not better than, the nativeenzymes for structural and functional studies. Third, biosyn-thetic models may offer additional advantages, since differentmetal-binding centers can be compared in the same proteinframework. Finally, the biosynthetic approach allows syn-thesis of new compounds, with structures and reactivitiesunprecedented in either biology or inorganic chemistry.

2. Application of Biosynthetic Inorganic Chemistryin Biomimetic Model Systems

Biomimetic model systems combine the benefits of bothchemistry and biology. In this process, one can learn theminimal structural features necessary to form metal-bindingsites with desirable function and help to elucidate thestructure–function relationship of target proteins. At thesame time, new compounds with structures that are rare ininorganic chemistry may be synthesized, which expands ourknowledge of coordination chemistry. The resulting com-pounds are often smaller and better defined structurally thanthe native proteins, and can therefore be used in practicalapplications.

Biosynthetic model systems with small, stable, and well-characterized proteins as ligands can achieve the same goals.Since they use the same ligands as target proteins and operateunder almost the same physiological conditions, they canoften result in closer models and are more readily usable inpractical applications. A few examples will illustrate thesefeatures.

2.1. Inorganic Biosynthesis with the Same Protein Scaffold andLoop-Directed Mutagenesis

The CuA center is a mixed-valence dinuclear coppercenter in which the copper ions are each coordinated to ahistidine residue (His181 and His224) and bridged by thethiolate sulfur atoms of two cysteine ligands (Cys216 andCys220, Figure 2A, top).[18–21] Weak ligands such as the

thioether sulfur atom of methionine (Met227) and thebackbone carbonyl oxygen atoms of isoleucine (Ile180),histidine (His224), and glutamate (Glu218) are also presentin axial positions (Figure 2A, bottom). The CuA center isunique in both inorganic chemistry and biology; it acts as anelectron-transfer center in cytochrome c oxidase (CcO; aterminal oxidase in the respiratory chain of eukaryoticmitochondria and some aerobic bacteria)[22–26] and nitrousoxide reductase (N2OR; an enzyme responsible for thereduction of N2O in denitrifying bacteria).[27, 28] It was alsothe first biological system shown to contain a metal–metalbond.[29] Many coordination compounds containing metal–metal bonds are known with late transition metals; howeverfew contain first-row transition metals such as copper.[30]

Whereas dinuclear or multinuclear mixed-valence coppercomplexes in which the unpaired electron is completelylocalized (Class I) or partially delocalized (Class II) havebeen known for many years,[31,32] copper complexes, such asthe CuA center, with fully delocalized (Class III) mixed-valence states are rare.[33–40] In both cases, the synthesis of theCuA center and elucidation of the structural features respon-sible for its functions are of great interest.

A number of synthetic models with organic ligands havebeen reported for this purpose, most of which contain adinuclear mixed-valence copper center and a copper–copperbond, but not bridging thiolate units.[33, 34,38,39,41] A modelcompound that contains bridging thiolate residues but lacks acopper–copper bond also exists.[36] A dinuclear copper centercontaining a copper–copper bond and two bridging nitrogenatoms was also reported recently.[42] Stable, easy-to-make, and

Figure 2. Crystal structures of A) the CuA center in cytochrome coxidase from P. denitrificans; and B) a biosynthetic CuA model inazurin. The top figures show the CuA center with Cu2S2(Cys) in a planeand the bottom figures show the CuA center viewed perpendicular tothe plane.


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5591Angew. Chem. Int. Ed. 2006, 45, 5588 – 5601 � 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org


well-characterized blue copper proteins such as amicyanin[43]

and azurin[44] were used as ligands to make a closer model ofthe CuA center. Blue copper proteins and proteins containingthe CuA center have been shown to share close structuralhomology.[45] Sequence alignment revealed that mononuclearblue copper proteins and dinuclear CuA proteins differ mainlyin a single loop between two b-strands. By replacing the loopin blue copper proteins with the corresponding loop in CuAproteins (Figure 3), a close mimic of the CuA center was

synthesized in blue copper proteins amicyanin[43] andazurin.[44] A high-resolution crystal structure showed thatthe CuA center in the azurin model is almost identical to thatof the native CuA center, including structural features in thesecondary coordination sphere, such as the nonbondinginteractions between the thioether group of methionineresidue and peptide carbonyl oxygen atoms (Figure 2B).[46]

The synthesis of a close model of a native protein is onlyone measure of the effectiveness of such an approach. Howmuch insight is gained into the structure and function of themetal center is another important measure. The biosyntheticCuA model also gives deeper insight in this regard. Forexample, a stopped-flow study of copper incorporation intometal-free CuA azurin revealed a tetragonal intermediate andshowed the importance of reductants in the formation of thefinal CuA center.

[47] Titration of different metal ions into themodel protein suggests that the CuA center as an M

IIM’ I

center (M, M’= any metal) is strongly favored regardless ofthe sequence of addition of metal ions.[48,49] Mutagenesisstudies revealed that the conserved cysteine,[50] histidine,[51,52]

and methinione[53] residues all play an important role inmaintaining the structure and in fine-tuning the function.Surprisingly, the dinuclear copper structure remains largelyintact, even when the histidine was replaced with residuesincapable of coordinating to the copper (e.g. Ala and Gly), asevidenced by the similar characteristic purple color, as well asUV/Vis, magnetic circular dichroism (MCD), resonanceRaman (RR), and electron nuclear double resonance(ENDOR) spectra.[51,52]

Until recently, there were not many examples of fullydelocalized (Class III) dinuclear mixed-valence copper com-pounds. Even rarer is the reversible conversion betweendifferent classes, that is, between delocalized [Cu1.5···Cu1.5]and trapped valence [CuII···CuI] states. The study of the CuA

center in azurin provided an interesting example of areversible transition between delocalized and trappedmixed-valence states that is triggered solely by a change inpH value.[54] The CuA center is known to be the electron entrysite in cytochrome c oxidase. However, its role in proton-coupled electron transfer has not been defined clearly. Thiswork showed that protonation of the C-terminal His residuenot only resulted in a trapped valance state of the CuA center,but also caused a significant increase in the reductionpotential.[54] Since the corresponding C-terminal His residuein cytochrome c oxidases is located along a major electron-transfer pathway from CuA center to heme a, and sinceprotonation can result in an increased reduction potential thatprevents electron transfer from the CuA center to heme a, theresults strongly indicate that the CuA center and the histidineresidue may play important roles in proton-coupled electrontransfer.[54]

These observations may be obtained from studies ofnative CuA centers, in particular soluble fragments of CcOcontaining the CuA center.

[18–21] However, several studies havedemonstrated that biosynthetic models can provide uniqueinsights that are not readily obtainable from studies of eithernative enzymes or synthetic model compounds. For example,by using azurin as the “ligand” and replacing the ligand loopof the blue copper center with that of the CuA center, twoelectron transfer (ET) centers may be placed in the sameprotein framework. High-resolution crystal structures showedthat one of the copper atoms in the CuA center overlaysexactly with the blue copper center (Figure 4A).[46] Therefore,

this biosynthetic model provided a unique opportunity tocompare the two ET centers in the same protein frameworkand exclude or at least minimize other factors that influenceits properties. For example, the same series of mutations at aconserved axial methionine unit in the blue copper protein(Met121, Figure 4B) and the CuA azurin (Met123, Fig-ure 4C) indicate that the methionine residue has much lessinfluence on the reduction potential of the CuA center(< 25 mV) than of the blue copper center (> 170 mV).[53] Incontrast, electron transfer from the same donor and at thesame distance to the CuA center is faster, despite its lowerreduction potential, than to the blue copper center.[55] Thus,this work showed directly that the CuA center is a moreefficient ET center.

Figure 3. Development of a biosynthetic CuA model in azurin throughloop-directed mutagenesis.

Figure 4. A) Overlay of the crystal structures of blue copper azurin (inlight blue) and the biosynthetic CuA model in azurin (in purple). Thecopper site in the blue copper center (B) overlays almost exactly withone of the copper sites in the purple CuA center (C).

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These findings may be important in understanding thedifferent roles of the two copper centers. A much wider rangeof reduction potentials (> 600 mV) is required for bluecopper proteins to transfer electrons to a variety of partnersin many different biological systems.[20, 21,56,57] In contrast, theCuA center is part of cytochrome c oxidase, which is at the endof the respiration chain with very small differences (< 50 mV)in the potentials of the redox partners.[58] In this case, largevariations in the potential could hinder the flow of electronsin the right direction. The diamond-shaped Cu2S2(Cys) corestructure of CuA provides a way to minimize changes inreduction potential through variations of the axial ligands.Despite the low driving force, however, the CuA center stillneeds to transfer electrons to its partners at a desirable rate.Therefore, it has to optimize its structure by lowering itsreorganization energy to be a more efficient ET center thanthe blue copper center.

Loop-directed mutagenesis, in which ligand loops of atemplate protein are swapped with those of a target proteinthat shares similar structural homology, was employed tomake the biosynthetic models of the CuA center describedabove. A preliminary survey of the protein structure databank (PDB) suggests that this approach is not limited tospecific cases but can be utilized generally to make biosyn-thetic models of many metalloproteins. For example, azurin,amicyanin, and CuA proteins belong to a group with acommon scaffold, called the Greek Key b-barrel. It is thesecond most abundant motif in the PDB, and a number ofproteins with diverse active-site structures and functions, suchas immunoglobin, beta-amylase, cytochrome c oxidase, nitritereductase, and superoxide dismutase, contain the samescaffold (Figure 5).[59]

Most of the active sites in these proteins reside within orbetween loops. Evidently, nature produced a thermodynami-cally stable scaffold upon which many different proteinstructures and functions can be conferred by simply swappingloops, which is much less disruptive to the protein structurethan changing scaffolds. This “trick” is important for biosyn-thetic inorganic chemistry.

Another example of nature using the same proteinscaffold for different functions are heme peroxidases.[60] All

heme peroxidases contain a heme active site. However,manganese peroxidase (MnP) contains an additional MnII-binding site (Figure 6A). MnII oxidation plays a critical role inthe function of MnP (Scheme 1) in the biodegradation oflignin, the second most abundant biopolymer on earth (after

Figure 5. The Greek Key b-barrel motif appears in more than 600proteins of different classes with diverse functions. As most of theactive sites are in the loop region, loop-directed mutagenesis can beused to construct novel metalloproteins.

Figure 6. A) Comparison of manganese peroxidase (MnP) and cyto-chrome c peroxidase (CcP); B) Heme-manganese binding site in MnPand CcP; C) Crystal structures of MnP (red) and a CcP variant thatmimics MnP (blue).

Scheme 1. Catalytic cycles of CcP (top) and MnP (bottom). Trp= tryp-tophan; Por=heme porphyrin.


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cellulose), as well as being a key step in converting woodyplants to small chemicals such as those in petroleum.[61]

Interestingly, cytochrome c peroxidase (CcP) shares notonly the same overall structure, but also a similar structureof the heme-binding site, the major differences being that CcPlacks the MnII-binding site and contains Trp instead of Phearound the heme-binding site (Figure 6A,B).[62] Severalligands to MnII in MnP are absent in CcP. To convert CcP,an enzyme found in bakerIs yeast, into an MnP with similarfunction, the residues in CcP were replaced with the residuesof MnP at the corresponding positions to give new CcPvariants with enhanced MnP function (Figure 6C).[63–65]

Furthermore, replacing the Trp residues in CcP with thePhe units from MnP showed that the Phe residues playdifferent roles in MnP function; whereas the Trp191Phemutation stabilized compound I (an intermediate), theTrp51Phe mutation increased the activity significantly, as itincreased the reactivity of compound II, whose oxidation ofMnII is the rate-determining step in the reaction mechanism ofMnP.[66,67]

2.2. Noncovalent Tuning of the Secondary Coordination Spherethrough Inorganic Biosynthesis

The noncovalent tuning of the secondary coordinationsphere of metal-binding sites is another “trick” of biologicalsystems. For example, cytochrome P450 (Cyt P450) is one ofthe most efficient enzymes for asymmetric oxygen-transferreactions, such as sulfoxidation, epoxidation, and hydroxyl-ation, of a variety of substrates.[68] Its primary coordinationsphere consists of a heme unit with a thiolate Cys as itsproximal ligand (Figure 7A). In view of the importance of

heme–thiolate ligation, the proximal ligand His in humanmyoglobin was changed to a Cys.[69,70] The spectrum of theresulting protein was similar to that of Cyt P450. The proteinmodel also exhibited increased asymmetric sulfoxidationactivity. However, the same His-to-Cys mutation alone inhorse heart myoglobin (Mb) did not result in heme–thiolateligation.[71] Instead, an additional mutation of the distalnoncoordinating histidine to either a valine or isoleucineresulted in a P450-like protein with an iron(III) resting state(Figure 7B).[71] This effect, termed the trans effect by theauthors, mimics the more hydrophobic environment of theCyt P450 (Figure 7A) and contributes significantly to thebinding of the axial ligand and to the stability.

Similarly, mutation of the axial His ligand to Cys in CcPresulted in a very unstable ligand that was rapidly oxidized tocysteic acid.[72] It was then recognized that a nonpolar residuenext to the Cys unit is conserved in P450 proteins, while theanalogous amino acid in CcP is an aspartic acid (Figure 7C).Thus, further mutation of this Asp residue to Leu resulted in astable, pentacoordinate, high-spin heme with thiolate liga-tion.[73] The study also marked the first time a stablecyanidoferric complex and the ferrous state of a model P450was obtained.[73,74]

Stable coordination of cysteine thiolate in the ferrouscarbonyl derivative of an engineered protein was alsoobtained by mutations of the axial His and Met ligands incytochrome b562 to Cys and Gly, respectively (Figure 7D).

[75]

The key to success was the replacement of two glutamateresidues (Glu4 and Glu8), which are electrostatically repel-led from the heme propionate groups, with Ser. These studiesdemonstrated the importance of the secondary coordinationsphere around the primary coordination ligands in stabilizingmetal–ligand ligation.

2.3.Water and Hydrogen Bonding in Inorganic Biosynthesis

The third “trick” emerging from recent investigations on anumber of metalloproteins is the precise positioning ofhydrogen-bonding networks in fine-tuning the function andspecificity of metalloproteins. For example, it has been shownthat most heme enzymes go through a similar heme–peroxointermediate and yet exhibit diverse enzymatic activities, suchas monooxygenase (Cyt P450), heme oxygenase (HO), andoxidase (CcO). High-resolution crystal structures of both CytP450 and heme oxygenase revealed the presence of watermolecules in the metal-binding sites (Scheme 2).[76,77] Thenumber and exact location of the water molecules have beenshown to be critical in forming hydrogen-bonding networksthat can dramatically influence the reactivity. Although theexact nature of the hydrogen-bonding networks in CcO hasnot been revealed, functional studies of a biosynthetic modelin myoglobin support their importance in fine-tuning thereactivity.[78] This work, complementary to work carried outusing synthetic models,[79, 80] is based on a structural compar-ison of myoglobin and heme–copper oxidase. Although bothproteins contain heme, CcO contains a copper center that isapproximately 4.7 J from the heme iron center (Figure 8A).To make a biosynthetic model of CcO, His ligands were

Figure 7. Key structural differences of Cyt P450 (A), Mb (B), CcP (C),and Cyt b562 (D).

Y. LuReviews



introduced into the distal side of sperm whale Mb at thecorresponding locations (Figure 8B).[81]

Spectroscopic studies indicate that the Mb model(CuBMb) is very similar to CcO. Studies of this biosyntheticmodel also indicated that the presence of a copper ion has acritical influence on the redox potential of the heme Fecenter[82, 83] and can transform the oxygen-binding protein Mbinto an oxygen-activating enzyme.[78,81] Interestingly, theprotein model in Mb generates verdoheme, a key intermedi-ate in HO, rather than producing a ferryl–heme as in CcO.[78]

Control experiments indicated that protein instability oraltered dynamics of the binding site were not the cause forthis reaction, since replacement of CuII or CuI by redox-inactive ions such as ZnII and AgI, respectively, does notpromote the HO reaction.[78] Since all reactions were carriedout in the presence of catalase, the possibility of exogenousperoxide as the cause for verdoheme formation was alsoeliminated. More importantly, reaction of CuBMb with H2O2

(equivalent in oxidation state to 2e�-reduced O2 but possess-ing two extra protons) produces a ferryl species similar to thatin CcO. Since HO results in verdoheme regardless of thepresence of metal ions in the distal site, or through reaction

pathways of either reduced heme with O2 or oxidized hemewith H2O2, the biosynthetic model CuBMb is not simply anHO model. Rather, CuBMb is at a branching point betweenHO and CcO (Scheme 3); the extra proton may promoteeither the CcO or the P450 reaction, but not the HO reaction.Therefore, such a biosynthetic model provides a uniqueopportunity to investigate which properties direct the enzymeactivity.

3. Biosynthetic Inorganic Chemistry for theSynthesis of New Inorganic Complexes

Biosynthetic inorganic chemistry has been quite effectivefor making biomimetic models of complex metalloproteinsand for revealing new insights into the structural featuresresponsible for protein function. Its major advantage, how-ever, is that it combines the benefits of inorganic chemistryand biology to produce new inorganic complexes withunprecedented structure and function.[84]

Natural metalloproteins normally consist of 20 aminoacids, less than half of which can bind as ligands to metal ions.Furthermore, they employ only few metal ions and metal-containing prosthetic groups (e.g., cobalamins and hemegroups). The use of nonnatural amino acids in metalloproteinswill dramatically increase the number of possible liganddonor sets, and the introduction of inorganic compounds intoproteins will lead to new and complex active sites. The netresult will be a new, considerably expanded enzymology,which can draw from a large reservoir of artificial metal-loproteins for various reaction types and rates.

Moreover, such endeavors can also result in new inorganiccompounds with novel structure and activity. Especiallyimportant in this respect is the adaptation of inorganiccatalysts for asymmetric reactions and for chemical trans-formations in water. Encapsulating these inorganic catalystswith proteins can fulfill both purposes, as the natural chiralenvironment of a protein can provide chiral discrimination.The hydrophobic interior of proteins provides a suitablesurrounding for inorganic complexes that are active only in

Scheme 2. Proposed role of hydrogen bonds in HO and cyt P450.Adapted from reference [76].

Figure 8. A) Overlay of crystal structures of CcO (thick lines) withsperm whale Mb (thin lines). Mb lacks the CuB-binding site since ithas one His, one Leu, and one Phe residue instead of three histidineresidues as in CcO; B) Overlay of the crystal structure of CcO (thicklines) with a computer model of CuBMb (Leu28His/Phe43Phe, thinlines).

Scheme 3. Hydrogen bonds and protons influence the reaction path ofthe common intermediate in heme enzymes. Adapted from refer-ence [78].


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organic solvent, while the hydrophilic exterior can increasethe water solubility. These requirements can all be accom-plished with minimal modification of the ligands and thusminimal effect on the catalyst reactivity.

3.1. Introducing Nonnatural Amino Acids into Metalloproteins

The properties of metalloproteins can be fine-tuned byintroducing nonnatural amino acids into the metal-bindingsites. For example, pyridine is a common ligand in syntheticmodels of metal-binding sites containing imidazole histidinesin proteins.[10] The histidine ligands have been replaced bypyridine ligands in a newly designed heme protein with foura-helix units. The replacement of the two His ligands with twopyridyl ligands decreased the protein-binding affinity of ferricheme approximately 60000-fold and increased the reductionpotential of the heme by 287 mV.[85] These differences shouldbe taken into consideration when using pyridines to modelhistidines in metalloproteins.

Perhaps the biggest advantage of using nonnatural aminoacids in the metal-binding sites of proteins is the isostructuralreplacement of ligands or residues around the secondarycoordination sphere, by which the electronic properties canbe changed through substitution of a single atom or group.For example, both Cys and Met in blue copper proteinproteins are conserved (Figure 9). Replacing the cysteine with

selenocysteine in azurin resulted in marked changes in theUV/Vis and EPR spectra (50-nm red shift of the visiblecharge transfer band and twofold increase in the hyperfinecoupling constant),[86, 87] with little effect on the reductionpotential of the copper center.[86] In contrast, replacing themethionine with selenomethionine or norleucine resulted inlittle change in the UV/Vis and EPR spectra, but a dramaticincrease in the reduction potentials (25 and 140 mV, respec-tively, over the native protein).[88]

In addition to ligands in the primary coordination sphere,residues in the secondary coordination sphere have also beenreplaced with nonnatural amino acids. For example, replace-ment of the OH group at the para position of a conserved Tyrunit near one of the Cys ligands of a rubredoxin with H, F,NO2, and CN groups resulted in an increase in reductionpotentials of the iron–sulfur center, with electron-withdraw-ing groups leading to more positive potentials (Fig-

ure 10A).[89] Furthermore, a conserved hydrogen bondbetween the backbone amide groups and the cysteine ligandof the Fe4S4 clusters was found in high-potential iron–sulfurproteins (HiPIPs). Replacement of the backbone amidegroups with an ester linkage eliminated these hydrogenbonds and lowered the reduction potential of the iron centerby approximately 100 mV (Figure 10B).[90]

Isostructural replacement of ligands has been a commonpractice in synthetic inorganic chemistry. Introducing non-natural amino acids into metalloproteins finally allowsbiosynthetic inorganic chemistry to achieve a similar levelof sophistication. Since proteins can provide a rigid networkfor the metal-binding sites, isostructural replacement oftenallows the introduction of ligands with little or no ability tocoordinate to metal ions (e.g., replacement of Met with Ile)without affecting the overall characteristics of the metal-binding site. It also makes it easier to probe noncovalentinteractions in the secondary coordination sphere (e.g.,through replacement of a peptide bond with an ester link-age[90]). This approach can reveal the role of specific residuesto an unprecedented level; it can be used to fine-tunecoordination complexes. For example, the use of isostructuralnonnatural amino acids at the Met position allowed decon-volution of different factors that influence the reductionpotential of the blue copper azurin through this axialligand.[88] A linear relationship between the reduction poten-tial and the hydrophobicity of the side chains on the axialligand pointed to hydrophobicity as the dominant factor incontrolling the reduction potential[88] and could be helpful indeveloping similar compounds with predicted reductionpotentials.

3.2. Introducing Nonnative Metal Cofactors into Proteins

The introduction of nonnative metal cofactors intoproteins exemplifies the combination of the benefits of

Figure 9. Isostructural replacement of Cys and Met by nonnaturalamino acids.

Figure 10. Using nonnatural amino acids to probe the role of Tyr inthe secondary coordination sphere in rubredoxin (A) and the backbonecarbonyl oxygen atom in HiPIP (B).

Y. LuReviews



biology and inorganic chemistry in biosynthetic inorganicchemistry. Three approaches have been applied towards thisgoal: noncovalent, single-covalent, and dual-covalent attach-ment.

The majority of native metal cofactors or prostheticgroups are incorporated into proteins through noncovalentinteractions such as hydrogen bonding, as well as electrostaticand hydrophobic interactions. Because proteins have notbeen evolved to bind nonnative metal cofactors specifically,the introduction of these cofactors is a challenging task. Themost successful approach thus far involves the replacement ofthe heme unit in heme proteins such as myoglobin withmodified metalloporphyrins.[91–99] Since the modified metal-loporphyrins are similar in structure to native heme groups, itis possible to confer novel reactivities to the protein throughthe introduction of new functional groups without severelychanging the structure of the metal-binding site. For example,modifications to the two propionate groups in heme resultedin new functions such as protein–protein and protein–molecule recognition as well as electron-transfer propertiesand enhanced chemical reactivity (Figure 11A),[99] whichmanifests itself for example in an enhanced P450-likedioxygen activation upon attachment of a flavin to one ofthe propionate groups.[102] Furthermore, when the heme isreplaced with iron porphycene,[103] enhanced O2-bindingaffinity is observed.

Many nonnative metal complexes do not resemble nativemetal cofactors. Conjugates of these metal complexes withbiotin, however, can take advantage of the strong and specificbinding between biotin and the protein avidin. In this way,biotinylated dirhodium(I) complexes have been introducedinto avidin or the closely related protein streptavidin toproduce novel catalysts for asymmetric hydrogenation withup to 96% enantiomeric excess (ee, Figure 11B).[100,104–111]

Another way to incorporate nonnative complexes is thedesign of metalloproteins based on the crystal structures ofproteins and metal complexes. After careful inspection of thestructures of myoglobin and MIII(salophen) complexes (M=

Cr, Mn), modifications of both the protein and the metalcomplex gave a new protein that exhibited asymmetricsulfoxidation activity (Figure 11C; salophen=N,N’-bis(sali-cylidene)-1,2-phenylenediamine dianion).[101, 112–114] Finally, amimetic protein with four helices has been designed de novothat selectively binds a metalloporphyrin.[115] This examplemarks the beginning of computationally developed, tailor-made metalloproteins with nonnative metal complexes.

Noncovalent attachment is not the only way in whichnative metal cofactors can be incorporated into proteins.There are also covalent attachment strategies, in whichattachment is through either a single point (some protozoanmitochondrial cytochrome c[116]) or two points (most cyto-chrome c[117]). These strategies have also been applied toincorporate nonnative metal complexes.

For example, the introduction of 1,10-phenanthrolinecopper[120] or iron–edta[121] into proteins by single-pointcovalent attachment gave a sequence-specific nuclease(edta= ethylenediaminetetraacetate). Covalent attachmentof a copper(II) 1,10-phenanthroline complex to a singlecysteine residue in an adipocyte lipid-binding protein gave a

catalyst that promotes highly enantioselective hydrolysis(with up to 86% ee) (Figure 12A).[118,122] This strategy wasalso used to attach a ferrocene derivative covalently to azurin(Figure 12B). This novel organometalloprotein increased thesolubility of ferrocene and improved the stability of ferroce-nium in water.[119] The secondary coordination sphere of the

Figure 11. Introducing nonnative metal-containing cofactors into pro-teins through noncovalent bonds. A) Replacing heme in Mb with amodified heme (adapted from reference [99]); B) Introducing organo-metallic complexes by taking advantage of strong noncovalent biotinand avidin interactions (adapted from reference [100]); C) Replacingheme in Mb with an Mn Schiff base complex (adapted from refer-ence [113]).


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protein has also been used to fine-tune the reduction potentialof ferrocene.[119]

Although single-point attachment allows the binding ofnonnative metal complexes to proteins with minimumstructural modifications, the conformational freedom of thecomplexes inside the protein may not be restricted enough toperform highly stereo- and enantioselective transformations.For example, an Mn(salen) complex coupled by single-pointcovalent attachment to a protein gave less than 10% ee(salen=N,N’-bis(salicylidene)ethylenediamine dianion).[123]

When the complex is attached in a specific location of Mbby a dual-point covalent attachment, the enantioselectivityincreased to 51% ee (Figure 13).[124] Clearly, such enantiose-lectivity is not yet useful for synthetic transformations;however, it is understandable given the fact that myoglobinhas not evolved like other heme enzymes (e.g., Cyt P450) forasymmetric catalysis, and that the substrate binding site hasnot been modified to confer substrate selectivity. Theprinciple demonstrated here is nonetheless important inguiding future research. For example, a combination ofthese covalent attachment strategies[123,124] with structure-based design or with directed evolution methods[125,126] willresult in the next generation of asymmetric catalysts witheven higher efficiencies and selectivities.

4. Conclusions and Outlook

Biosynthetic inorganic chemistry is a natural extension ofsynthetic inorganic chemistry. Instead of using small organicmolecules, small, stable, easy-to-make, and well-characterizedproteins such as azurin and myoglobin are used as “ligands”for the synthesis of either biomimetic models or newcompounds. The chemical and biological approaches eachhave advantages and disadvantages in terms of synthesis,characterization, and properties (Table 2).

Synthesis. A synthetic approach is generally faster andproduces higher yields. However, recent developments inmolecular biology and protein enzymology have enabled theroutine production of hundreds of milligrams to grams ofprotein in the laboratory and even higher yields withindustrial methods. By carrying out reactions with cellscontaining the desirable protein models, the power of naturalamplification of genes and protein products can be exploitedfor biosynthesis and biocatalysis. One advantage of biosyn-thesis over small molecule synthesis, in which modification ofthe ligand might result in significant variations in yield andhave associated cost and time investments, is that a similarmodification of a protein ligand has a smaller influence onthese parameters. These features make the speed and yield ofbiosynthesis closer to that of small molecule synthesis. In afew selected cases, it is even easier to synthesize biosyntheticmodels than some of the more complex synthetic models. Forexample, synthetic models of heme–copper oxidases requiremultistep syntheses that give relatively low yields.[79, 80] Thebiosynthesis of heme–copper models in myoglobin[81] orcytochrome c peroxidase[127] proceeds with similar yields andpreparation times as biosynthesis of other derivatives of thesame proteins. Regardless of the complexity of the modifica-tions, approximately 100 mgL�1 of pure biosynthetic modelscan be obtained in less than a week. The routine production ofgram quantities of protein is thus possible. Although thebiosynthetic approach has been limited to a few cases inindustry,[13, 14] the gap between the two approaches is narrow-ing.

Figure 12. Single-point covalent attachment of nonnative metal-con-taining cofactors to proteins. A) A computer model of an adipocytelipid-binding protein–phenanthroline complex (ALBP-Phen) (adaptedfrom reference [118]); B) A computer model of an azurin–ferroceneorganometalloprotein.[119]

Figure 13. Introducing nonnative metal-containing cofactors into pro-teins through dual-point covalent attachment. A computer model ofMb(L72C/Y103C) with a MnIIIsalen complex covalently attached at twopoints and overlaid with heme in myoglobin (adapted from refer-ence [124]).

Y. LuReviews



Applications. Biosynthesis is often carried out in air atambient temperatures and pressures rather than in inertatmospheres at extreme temperatures and pressures, as arerequired for many syntheses of small molecules. Therefore,biosynthesis could help to save energy and equipment costs.Furthermore, biosynthesis is an environmentally benignapproach, since biodegradable materials are utilized andwater is the solvent. However, the products from biosynthesesare not as robust as synthetic compounds, which will make thelong-term industrial use at high temperatures or pressuresdifficult. This problem can be overcome by using proteinligands that have been isolated from extremophilic organisms(which live at high temperatures or in organic solvents such asmethanol).

A common criterion to evaluate the practical utility ofcatalysts is the mass ratio between the product and thecatalyst. This is a good measure only if the catalysts are ofsimilar type and are synthesized by similar methods. In thiscase, higher mass catalysts are generally more costly tosynthesize and to dispose of, and therefore smaller com-pounds would be preferable to a chemically synthesizedprotein ligand of similar catalytic efficiency. However,biologically synthesized protein ligands can almost alwaysbe made at a much lower cost than their molecular weightwould suggest, and disposing a biocompatible and biodegrad-able protein ligand is also much less costly than disposing asimilarly sized organic ligand, which is often not biocompat-ible or biodegradable. In fact, the cost of synthesizing anddisposing of a small protein (e.g., MW= 10000) by usingrecombinant protein expression systems is very similar to thatof a much larger protein (e.g., MW= 100000). Therefore,multiple criteria are needed to evaluate different types ofcatalysts,[128] for example, mass ratio, catalyst accessibility andcosts of synthesis and disposal, substrate scope, and, ifapplicable, enantioselectivity.

Characterization. Most metal-based spectroscopic techni-ques, such as electronic spectroscopy (UV/Vis), electronparamagnetic resonance (EPR), magnetic circular dichroism(MCD), resonance Raman (RR), and X-ray absorptionspectroscopy (XAS), can be performed on both syntheticand biosynthetic compounds without much difference in datacollection and interpretation. Even though recent develop-ments in structural biology have made it possible to obtainthree-dimensional structures of biosynthetic compoundsroutinely by using X-ray crystallography or NMR spectros-copy, it is still much easier to obtain structures of syntheticcompounds with higher resolution. To overcome theselimitations, one can use proteins with known 3D structuresas ligands. In this way, it is easier to grow diffraction-qualitycrystals, and 3D structures can be obtained faster by compar-ing differences in the electron density map. The use ofsynchrotron sources to obtain diffraction data may also helpto improve resolution.

Properties. Proteins provide a rigid network that helps tostabilize metal-binding or substrate-binding sites as a result ofcovalent bonds and noncovalent interactions (e.g., hydrogenbonds, electrostatic and hydrophobic forces) in the primaryand secondary coordination spheres. They provide a betterenvironment for regio- and stereoselective binding and

catalysis, often without requiring protection of reactivefunctional groups. Because they consist of chiral naturalamino acids, proteins are intrinsically enantioselective. Bio-synthetic compounds with proteins can benefit from thesefeatures. Indeed, a number of biophysical studies show thatbiosynthetic models, such as a biosynthetic CuA center inazurin (Figure 2B),[46] are as rigid as the native templateproteins. Moreover, most synthetic modeling results in eitherstructural or functional models of target native proteins.Because biosynthetic models use the same type of ligands andare synthesized and characterized under the same conditionsas target proteins, they are often structural and functionalmodels at the same time. Recent advances in syntheticchemistry have made it possible to design new organic ligandsthat provide a rigid network similar to that in proteins,including hydrogen-bonding interactions in the secondarycoordination sphere.[11,12] Synthetic asymmetric catalysts havea wider range of activities and a broader substrate scope.[129]

The wider range of activities arises mainly from the extensivechoice of ligands, metals, and metal-containing cofactors. Thebroader substrate scope is attributable to a wide range ofaccessible reaction conditions, such as low temperatures, highpressures, and different organic solvents. Biosynthetic cata-lysts, however, are mostly restricted to only the 20 naturalamino acids, physiologically available metals or metal-con-taining cofactors, physiological conditions, and water assolvent. These limitations make biocatalysts even moreremarkable, and make it even more imperative to learnmethods from nature to make better catalysts. The introduc-tion of nonnatural amino acids and nonnative metal-contain-ing cofactors into proteins is an important step in thisdirection.

In summary, recent advances in a number of areas inbiology and chemistry have enabled the development ofbiosynthetic inorganic methods with comparable preparationtimes and yields to those of synthetic inorganic chemistry. Inmany cases, biosynthetic inorganic chemistry can produceclose structural and functional models of more-complexmetalloproteins and novel inorganic compounds. Thesedevelopments, however, only demonstrate the potential thatthe biosynthetic approach has to offer; this potential is farfrom being realized. Although methodologies have beendeveloped to introduce nonnative metal cofactors intoproteins by noncovalent, single-, and double-covalent attach-ment, the use of these approaches in asymmetrically catalyzedsyntheses of chiral intermediates for fine chemicals orpharmaceutical products is still lacking. In contrast to thefield of asymmetric catalysis, which has had many years ofdevelopment by a number of researchers, the field ofbiosynthetic inorganic chemistry is still in its infancy. Muchmore time and effort, including cross-fertilization of thesynthetic and biosynthetic approaches, will be required beforethe full potential of biosynthetic inorganic chemistry can berealized.

I wish to thank the members of my research group whose workhas been cited in the references for their dedication and hardwork, as well as Hee Jung Hwang, Thomas D. Pfister, DewainGarner, and Natasha Yeung for help with the preparation of


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figures in this review. The work of my group described in thisreview has been supported by the US National ScienceFoundation and the National Institutes of Health.

Received: January 16, 2006Published online: August 10, 2006

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