- Email: [email protected]
Protein engineering in bioelectrocatalysis
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Protein engineering in bioelectrocatalysis Tuck Seng Wong and Ulrich Schwaneberg Electrochemistry of redox proteins is a broadly applicable technology with important applications in biosensors, biofuel cells and chemical syntheses. Escalating attention in this area is driven by remarkable progress in designing efficient interfaces for transferring electrons between electrode surfaces and redox proteins. Research in interface design is slowly shifting from modifying electrode surfaces towards the engineering of redox proteins. Protein engineering, which encompasses rational design, directed evolution and combined methods, offers many powerful methods and strategies for improving the electron transfer properties of redox proteins. Addresses International University Bremen (IUB), Campus Ring 8, 28759 Bremen, Germany e-mail: [email protected]
Current Opinion in Biotechnology 2003, 14:590–596 This review comes from a themed issue on Chemical biotechnology Edited by Frances H Arnold and Anton Glieder 0958-1669/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2003.09.008
Abbreviations epPCR error-prone polymerase chain reaction HRP horseradish peroxidase GOx glucose oxidase LCCI laccase from Trametes versicolor
Introduction Nature has evolved a myriad of impressive proteins for shuttling electrons and catalyzing oxidative and reductive reactions. These redox proteins account for approximately one-quarter of all known proteins. In the field of bioelectrocatalysis, redox proteins are mainly used for biosensing. Emerging applications include biofuel cells for powering miniaturized implanted medical devices and chemical syntheses of pharmaceuticals and fine chemicals. Redox proteins are not evolved or optimized for applications in bioelectrocatalysis; they are fine-tuned to operate efficiently under the conditions of their native environment. Natural selection prevents random electron transfer in living organisms and ensures that valuable energy resources are not wasted and that toxic metabolites such as hydrogen peroxide are not produced. These goals are achieved by embedding redox-active Current Opinion in Biotechnology 2003, 14:590–596
centers deeply in an ‘insulating’ protein shell and by regulating electron transfer through sophisticated control mechanisms. Willner, Katz, Scheller and Schuhmann have summarized progress in using redox proteins for bioelectrocatalytical applications and have addressed design issues of electrode surfaces for efficiently relaying electrons to redox proteins in four excellent reviews [1,2,3,4]. In this review, we focus on protein engineering approaches for increasing electron transfer rates from electrode surfaces to redox proteins and discuss strategies for tailoring redox proteins to our needs in applications.
Electron transfer in redox proteins Electron transfer plays a pivotal role in biological functions essential to life, such as photosynthesis, respiration and metabolic pathways. In Marcus’ theory, three factors determine electron transfer within proteins [5]: reorganization energies (qualitatively reflecting the structural rigidity in the oxidized and reduced form); potential differences and orientations of involved redox-active sites; and the distances between redox-active sites and the intervening medium. The electrode and enzyme active site can be regarded as electron donor–acceptor pair. The protein or glycoprotein shell surrounding the active site becomes an effective kinetic barrier for heterogeneous electron transfer and can interfere with efficient direct electrical communication.
Recent advances in electrochemical applications of redox proteins Novel electrode surfaces that act as efficient interfaces for transferring electrons significantly broaden the repertoire of enzymatic reactions that are electrochemically accessible. Progress has been especially remarkable for P450 enzymes [6]. Recent highlights in biofuel cell research and chemical syntheses are summarized in Table 1. Additional innovations can be expected from fundamental research efforts, in which bioelectrochemistry is used as a tool for studying intermolecular biological electron transfer [7] and protein folding [8].
Challenges of bioelectrocatalysis employing redox proteins Being in its infancy, bioelectrocatalysis continues to be confronted with a broad range of challenges some of which are outlined below (Figure 1). Field conditions
Proteins exhibit high activities often at conditions close to their ‘natural’ environment. Glucose oxidase (GOx) for www.current-opinion.com
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Table 1 Bioelectrocatalytical highlights in biofuel cell research and chemical syntheses. Application
Examples
Novelty
Biofuel cell (anode/cathode)
Glucose oxidase/laccase Glucose oxidase/cytochrome c and cytochrome c oxidase Glucose oxidase/bilirubin oxidase Glucose oxidase/bilirubin oxidase Glucose dehydrogenase/bilirubin oxidase Glucose oxidase
High power output (0.78 V) [30] Switchable and tunable power output [31]
Interesting anodes
Glucose oxidase
Chemical syntheses (enzyme/application)
P450cam and myoglobin/styrene epoxidation P450cam/styrene epoxidation
instance, works well in a slightly acidic environment, but when employed in an implanted medical device its activity is compromised by the blood pH (7.3) and high chloride concentration (98–108 mM). Enzymatic properties Cofactor requirement
Cofactor regeneration poses a major challenge for many applications employing redox proteins. Cofactors such as NADH or NADPH are expensive and tend to be adsorbed at electrode surfaces. These adsorbed cofactors can subsequently be oxidized, leading to degradation of the cofactors and modifications of the electrode surfaces [9]. Enzyme instability
Maximum current output can often only be maintained for a short period of operation owing to ‘degradation’ of redox proteins and irreversible surface modifications [10]. Regarding aerobic cathodic systems, a major challenge is to run the reaction without reducing molecular oxygen to reactive species such as superoxide or peroxide that oxidatively damage the biocatalysts.
Power generation (2.4 mW, 0.52 V) with a grape [32] High power output (50 mW, 0.5 V) under physiological conditions [33] High power density (58 mW) under physiological conditions [34] A novel electrochemical communication. Gold nanoparticles act as an electron relay for the alignment of the enzyme on the conductive support and for the electrical wiring of its redox-active center [35] Increased electron diffusion coefficient by the tethering of redox centers to the backbone of the cross-linked redox polymer backbone through 13-atom spacer arms [36] Electrochemical-driven epoxidation of styrene with ultrathin polyion film [37] Styrene epoxidation in an electroenzymatic reactor [38]
provide linkages, and reduction of enzyme activity due to immobilization. Electron transfer mechanism in redox proteins
The electron transfer in redox proteins is tightly regulated to improve the fitness of the organism by avoiding waste of valuable energy resources and the generation of toxic intermediates. Regulation mechanisms within proteins are highly advanced (Figure 2) and well-gated on the molecular level by structural rearrangement within the proteins. For example, P450 BM-3 has a substrateinduced thermodynamic switch that regulates the electron transfer [12]. In the absence of substrate, no electron transfer from flavin mononucleotide (FMN) to heme occurs. This control mechanism prevents futile consumption of energy resources and avoiding the production of reactive and cell-damaging oxygen species such as hydrogen peroxide. Kinetic measurements for P450 BM-3 showed that the rearrangements within the protein for electron transfer, and not the oxygenation chemistry, are likely to be rate limiting. Conducting interface
Enzyme pretreatment
Oxidative pretreatments for cleaving the sugar moieties of redox proteins expressed in fungi and yeast are common approaches for boosting electron transfer rates by reducing the distance between redox proteins and electrode surfaces. However, strong oxidizing agents such as periodates often reduce enzymatic activity significantly [11]. Enzyme immobilization
Immobilization of redox proteins on electrode surfaces to achieve reproducible and fast electron transfer is often a trial and error process. Technical challenges include the even distribution of enzymes on electrode surfaces, the modification of enzymes or electrode surfaces to www.current-opinion.com
Electron transfer rates from redox proteins to electrode surfaces are key determinants of the overall performance and efficiency of bioelectrochemical devices. In the past few years, researchers have achieved remarkable progress in designing effective interfaces between redox proteins and conductive supports. Challenges encountered include electrode design, electrode surface functionalization for protein immobilization, irreversible adsorption and the degradation of cofactors, to name a few. Most approaches aim to optimize electron transfer rates by leaving the amino acid composition of the enzymes unchanged. Extensive research has been performed on mediated electron transfer [13], direct electron transfer between redox proteins and electrodes, electron transfer cascades via redox hydrogels, Current Opinion in Biotechnology 2003, 14:590–596
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Figure 1
Figure 2 Cytochrome P450 BM-3
Electrode NADPH
FAD
TC
FMN
Heme b
Fatty acid
Successful protein engineering strategies Nitric oxide synthase
Trimming the protein
NADPH
FAD
FMN
Heme b
L-Arg
Protein surface modification(s) TC
TC
Active-site mutation(s) L-Arg
Heme b
FMN
FAD
NADPH
Domain shuffling Cytochrome c oxidase Cyt c
CuA
Heme a
H+ Heme a /Cu 3 B
O2
Cytochrome cd1
e– Transfer Cyt c
Heme c
CG
Heme d1
NO2–
Current Opinion in Biotechnology
Field condition(s) (blood)
Four well-studied electron transfer chains in prominent redox proteins. Arrows indicate electron pathways and electron transfer is controlled by thermodynamics (TC, thermodynamic control), conformational gates (CG), and coupled electron/proton transfer (Hþ).
Enzyme properties (pH, temperature) Conducting interface design Understanding and tuning electron transfer within a redox protein
Challenges for protein engineering in bioelectrocatalysis Current Opinion in Biotechnology
Successes and challenges of protein engineering in bioelectrocatalysis.
and conducting polymer films [4]. Katz and coworkers [14] recently demonstrated an innovative approach for electrochemically driving oxidoreductases containing flavin adenine dinucleotide (FAD), or using NAD(P)þ. High bioelectrocatalytic activities were achieved especially with the GOx system, in which an apo-GOx (lacking its FAD) was reconstituted with an FAD cofactor that is covalently coupled to a phenylboronic-acid-functionalized gold electrode.
Protein engineering of redox proteins for electrochemical applications Protein engineering offers attractive solutions for overcoming physiological constraints. It improves enzymatic Current Opinion in Biotechnology 2003, 14:590–596
properties and enhances the electrochemical performance of bioelectronics by directing engineering focus on the redox protein, a key determinant in bioelectrocatalysis. Protein engineering technology has matured rapidly and many powerful genetic engineering tools are available [15]. General approaches in protein engineering include rational design, directed evolution (laboratory evolution), and combined efforts. Rational design
Rational design of proteins is driven by the hypotheses of researchers and computer models based on available crystal structures of the protein of interest. Site-directed mutagenesis is the method used in rational design for verifying deduced protein–function relationships. To rationally design redox proteins for biofuel cells and sensing applications several strategies have been used and are outlined below. Trimming the ‘fat’
The active site of an oxidoreductase is seldom found on the protein surface. By removing the amino acids that are not essential for maintaining protein function and structure, one can open up the active site and position a conducting support in close proximity to it. Two prominent examples in which this strategy was successfully used for improving electron transfer rates are microperoxidases and laccase from Trametes versicolor (LCCI). www.current-opinion.com
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Microperoxidase-11 is an undecapeptide (N-Val-GlnLys-Cys-Ala-Gln-Cys-His-Thr-Val-Glu) with a heme c group attached through thioester bonds at the two cysteine residues. It was obtained by enzymatic digestion of cytochrome c, and still exhibits electrochemical properties [16]. Microperoxidase-11 reveals several advantages over peroxidases in peroxide reduction: it is much smaller in size, exhibits high stability and direct electrical communication with many different electrodes because of its heme exposure, and has been successfully employed in biofuel cells. With laccase, Palmore and co-workers [17] expressed a truncated laccase LCCI in Pichia pastoris as the heterologous host. The amino acid sequence of this truncated laccase is identical to that of LCCI except that the final 11 amino acids (PIYDGLSEANQ; in single-letter amino acid code) at the C terminus of LCCI are replaced by a single cysteine residue. On the basis of their analysis of available crystal structures of laccase (Protein Data Bank [PDB] code 1a65 from Coprinus cinereus) and ascorbate oxidase (PDB code 1AOZ, a similarly structured copper oxidase from Cucurbia pepo medullosa), they hypothesized that the rate of heterogeneous electron transfer might increase if they widened access to the type 1 copper site of LCCI. This hypothesis was investigated by reducing the number of amino acid residues between the last histidine that binds the type 1 copper ion in the active site of LCCI (His478) and the C terminus (Gln519). Introduction of a cysteine residue at the C terminus of laccase provides a chemical target (i.e. thiol) for selective modification. Results from electrochemical studies showed that the redox potential of the active site of truncated laccase was shifted to more negative values than those found in other fungal laccases. Electron transfer from the electrode surface to the protein was observed for the truncated LCCI in contrast to no cyclic voltammogram recorded for the native enzyme.
ferrocenecarboxylic acid, to GOx for improving sensitivity and stability of glucose biosensors. The modified GOx showed comparable Km and kcat values to those of the wild-type enzyme; however, in the presence of ferrocenecarboxylic acid the polylysine GOx retained 90% of its activity, whereas native GOxs retained only 22% [20]. Halliwell and coworkers [21] introduced several tags in a L-lactate dehydrogenase (N- and C-terminal (poly)histidine tags and a C-terminal cysteine) enhancing the current density at the bioanode by a factor of 5 to 7 at certain lactate concentrations.
Protein surface modifications
Directed evolution
Protein surface modifications to achieve better heterogeneous electron transfer and cell performance have recently been reported. Gorton and co-workers studied the effect of His-tag and surface-exposed cysteine residues on the electrochemical properties of horseradish peroxidase (HRP). Non-glycosylated HRP containing a six-histidine tag at the C terminus, C(His)rHRP, was adsorbed on a pre-oxidized gold electrode and generated a more than 30-fold increase in electron transfer rate [18]. Introducing additional cysteine residues, preferably at the C terminus, led to a favorable direct proton-coupled or proton-gated electron transfer. The improved electron transfer rates were explained on the molecular level by surface interactions that play a dominant role in immobilizing and orienting HRP on electrode surfaces [19]. Another successful example was GOx, where a polylysine chain was added at the C terminus. The polylysine chain was added to anchor the electron transfer mediator,
The lack of a profound understanding of the relationship between protein structure and electron transfer hinders scientists from rationally designing efficient interfaces and pathways for transferring electrons (Figure 1). Directed evolution does not require knowledge of structures or functions and allows the tailoring of enzymes to our needs by mimicking Darwinian evolution [26]. Directed evolution will be very valuable in discovering the fundamental design principles of electron transfer in redox proteins when applied to proteins of known structure. In a directed evolution experiment, a pool of mutated enzymes is created via modification of the DNA and resulting proteins with beneficial properties are identified by selection or screening. This iterative process is repeated until the desired trait is improved. Random mutagenesis by error-prone polymerase chain reaction (epPCR) is most commonly used for creating diversity on the genetic level. The mismatches are achieved by
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Active-site mutations
Introducing mutations in the active site is an additional option for improving intermolecular electron transfer and substrate specificity, as shown by Davidson who studied the mechanisms of catalysis and electron transfer in methylamine dehydrogenase [22]. In this study, Phe55 of the a subunit, located where the substrate channel from the enzyme surface opens into the active site, was substituted with alanine. The engineered methylamine dehydrogenase (Phe55Ala) improved the sensitivity of a histamine sensor up to fourfold owing to the lower Km value [23]. Domain shuffling
A new enzyme engineering concept was introduced by Gilardi’s group who rationally constructed P450s that displayed improved electrochemical properties by assembling catalytic and electron transfer modules using ‘gene fusion’ techniques [24]. Domain shuffling is a widely used genetic engineering technique for swapping functional modules among closely related proteins. In addition, Gilardi’s group engineered a nucleic acid binding protein, rop, into a heme-binding protein that displays typical spectrophotometric and electrochemical properties of a heme protein [25].
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Table 2 Comparison of rational design and directed evolution. Aspect
Rational design
Directed evolution
Methods for mutating DNA Understanding of structure–function relationships Availability of crystal structure Key to success Experimental time for mutagenesis
Site-directed mutagenesis Important prerequisite
epPCR and recombination methods Not important
Important prerequisite A reliable model Short: single or few site-directed mutagenesis experiments 1–2 mutations
Not important A reliable high-throughput screening method Long: developing of a robust screening protocol depending on library size and iterative cycles With epPCR often 1–3 mutations
Determined by experimenters Determined by experimenters
Random Biased by employed polymerase and error-prone conditions
Number of mutations per mutagenesis experiment per gene Location of the mutation(s) Nucleotide substitution
employing DNA polymerase with low fidelity. The fidelity can further be reduced by addition of Mn2þ and/or an imbalanced nucleotide concentration [27]. Combining diverse genes that were selected for function by natural evolution can be an alternative method to epPCR for generating genetic diversity. Many powerful recombination methods, such as StEP, DNA shuffling, nonhomologous random recombination [28], SHIPREC, ITCHY and RACHITT, have been developed for molecular breeding [15]. Proteins will encounter operating conditions in electrochemical applications that strongly deviate from the physiological prerequisites (e.g. controlled electron flow, isolating protein shell) that natural selection imposes. For instance, heterogeneous electron transfer from redox proteins to electrodes is a property that has not been under selective pressure in nature. Accumulating new mutations by random mutagenesis may be more promising in ‘evolving’ this novel function than recombining proteins designed by nature that do not have the ‘trait’. However, DNA shuffling methods that introduce random mutations might be valuable alternatives. In a directed evolution experiment, a rapid screen or selection that reflects desired functions is the key to success. Enzymatic properties that are important for bioelectrocatalytical applications include reduced Km values for improving biosensors, increased tolerance towards field conditions (e.g. temperature, pH, inhibitors) and higher catalytic activities for boosting the power output of biofuel cells. Directed evolution has been proven a powerful tool for evolving all of these properties. An important lesson from directed evolution is that it has been especially easy to evolve traits that have never been under selective pressure in nature. Hence, it is encouraging to evolve heterogeneous electron transfer by directed evolution even though, at least not to our knowledge, it has not been reported yet. In our opinion, colorimetric or fluorometric assays based on electron transfer mediators that show color change upon Current Opinion in Biotechnology 2003, 14:590–596
a switch of oxidation state seem to be promising systems for directed evolution. A more costly, but also feasible, approach is to use potentiostats in a high-throughput format. Designing a protein engineering experiment requires weighing the pros and cons of rational design and directed evolution (Table 2) and needs the experimental constraints of the screening or selection system to be taken into consideration. Combining rational design and directed evolution (semi-rational design or rational evolution) often substantially accelerates the progress in protein engineering [29].
Conclusions Researchers are now borrowing a page from biology’s manual to create tailor-made proteins for applications in sensors, biofuel cells and syntheses of pharmaceuticals and fine chemicals. Despite the far-stretching potential in applications, there are considerable challenges in discovering fundamental structure–function relationships before we can understand and tune electron transfer. Rational design has proven its potential and many success stories in directed enzyme evolution have shown that it is especially easy to improve traits never required for biological function, such as stability or activity in a nonnatural environment or activity towards a non-natural substrate. This raises the hope that redox proteins can be engineered successfully, paving the way to a biotechnological revolution in biosensors, biofuel cells and biocatalysis.
Update Recent investigation showed that trimethylamine dehydrogenase mutant (Tyr422Cys) could be successfully immobilized on electrode surface that was chemically modified with redox polymer, enabling direct electrical communication between the enzyme and electrode [39]. Amperometric bi-enzyme sensor using NADH regeneration was also recently reported and this solves the fundamental difficulty in cofactor recycling [40]. www.current-opinion.com
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Acknowledgements We thank the Office of Naval Research (ONR) for support and Kang Lan Tee for checking the manuscript.
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14. Zayats M, Katz E, Willner I: Electrical contacting of flavoenzymes and NAD(P)R-dependent enzymes by reconstitution and affinity interactions on phenylboronic acid monolayers associated with Au-electrodes. J Am Chem Soc 2002, 124:14724-14735. This paper introduces a novel method for direct coupling of native cofactors such as FAD or NAD(P)þ, to an electrode surface. The enzyme electrodes are remarkably stable and no leakage of the cofactor was observed during operation. 15. Kurtzman AL, Govindarajan S, Vahle K, Jones JT, Heinrichs V, Patten PA: Advances in directed protein evolution by recursive genetic recombination: applications to therapeutic proteins. Curr Opin Biotechnol 2001, 12:361-370. This interesting review explains the principles of directed protein evolution and, in particular, advances in gene recombination methods. The pros and cons of each recombination method are considered. A complete list of recombinant protein therapeutics is included. 16. Katz E, Sheeney-Haj-Ichia L, Willner I: Magneto-switchable electrocatalytic and bioelectrocatalytic transformations. Chemistry 2002, 8:4138-4148. 17. Gelo-Pujic M, Kim HH, Butlin NG, Palmore GT: Electrochemical studies of a truncated laccase produced in Pichia pastoris. Appl Environ Microbiol 1999, 65:5515-5521. 18. Ferapontova E, Gorton L: Effect of pH on direct electron transfer in the system gold electrode-recombinant horseradish peroxidase. Bioelectrochemistry 2002, 55:83-87. 19. Ferapontova E, Schmengler K, Borchers T, Ruzgas T, Gorton L: Effect of cysteine mutations on direct electron transfer of horseradish peroxidase on gold. Biosens Bioelectron 2002, 17:953-963. 20. Chen LQ, Zhang XE, Xie WH, Zhou YF, Zhang ZP, Cass AEG: Genetic modification of glucose oxidase for improving performance of an amperometric glucose biosensor. Biosens Bioelectron 2002, 17:851-857. 21. Halliwell CM, Simon E, Toh CS, Cass AEG, Bartlett PN: The design of dehydrogenase enzymes for use in a biofuel cell: the role of genetically introduced peptide tags in enzyme immobilization on electrodes. Bioelectrochemistry 2002, 55:21-23. 22. Davidson VL: Probing mechanisms of catalysis and electron transfer by methylamine dehydrogenase by site-directed mutagenesis of aPhe55. Biochim Biophys Acta 2003, 1647:230-233. 23. Bao L, Sun D, Tachikawa H, Davidson VL: Improved sensitivity of a histamine sensor using an engineered methylamine dehydrogenase. Anal Chem 2002, 74:1144-1148. 24. Gilardi G, Meharenna YT, Tsotsou GE, Sadeghi SJ, Fairhead M, Giannini S: Molecular lego: design of molecular assemblies of P450 enzymes for nanobiotechnology. Biosens Bioelectron 2002, 17:133-145. An innovative approach to tailoring bioelectrochemical properties of redox proteins is presented by assembling catalytic and electron transfer ‘domains’ from closely related proteins. Examples of assembled proteins with enhanced bioelectrochemical performances are provided. 25. Wilson JR, Caruana DJ, Gilardi G: Engineering redox functions in a nucleic acid binding protein. Chem Commun (Camb) 2003, 7:356-357. 26. Glieder A, Farinas ET, Arnold FH: Laboratory evolution of a soluble, self-sufficient, highly active alkane hydroxylase. Nat Biotechnol 2002, 20:1135-1139. This outstanding paper describes how a fatty acid hydroxylating monooxygenase is converted into a powerful alkane hydroxylase and represents a remarkable example of directed evolution at its best. Alkane monooxygenases are of significant economical interest. 27. Cadwell RC, Joyce GF: Mutagenic PCR. PCR Methods Appl 1994, 3:S136-S140. 28. Bittker JA, Le BV, Liu DR: Nucleic acid evolution and minimization by nonhomologous random recombination. Nat Biotechnol 2002, 20:1024-1029. 29. Li QS, Schwaneberg U, Fischer M, Schmitt J, Pleiss J, Lutz-Wahl S, Schmid RD: Rational evolution of a medium chain-specific cytochrome P450 BM-3 variant. Biochim Biophys Acta 2001, 1545:114-121. Current Opinion in Biotechnology 2003, 14:590–596
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Protein engineering in bioelectrocatalysis Tuck Seng Wong and Ulrich Schwaneberg Electrochemistry of redox proteins is a broadly applicable technology with important applications in biosensors, biofuel cells and chemical syntheses. Escalating attention in this area is driven by remarkable progress in designing efficient interfaces for transferring electrons between electrode surfaces and redox proteins. Research in interface design is slowly shifting from modifying electrode surfaces towards the engineering of redox proteins. Protein engineering, which encompasses rational design, directed evolution and combined methods, offers many powerful methods and strategies for improving the electron transfer properties of redox proteins. Addresses International University Bremen (IUB), Campus Ring 8, 28759 Bremen, Germany e-mail: [email protected]
Current Opinion in Biotechnology 2003, 14:590–596 This review comes from a themed issue on Chemical biotechnology Edited by Frances H Arnold and Anton Glieder 0958-1669/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2003.09.008
Abbreviations epPCR error-prone polymerase chain reaction HRP horseradish peroxidase GOx glucose oxidase LCCI laccase from Trametes versicolor
Introduction Nature has evolved a myriad of impressive proteins for shuttling electrons and catalyzing oxidative and reductive reactions. These redox proteins account for approximately one-quarter of all known proteins. In the field of bioelectrocatalysis, redox proteins are mainly used for biosensing. Emerging applications include biofuel cells for powering miniaturized implanted medical devices and chemical syntheses of pharmaceuticals and fine chemicals. Redox proteins are not evolved or optimized for applications in bioelectrocatalysis; they are fine-tuned to operate efficiently under the conditions of their native environment. Natural selection prevents random electron transfer in living organisms and ensures that valuable energy resources are not wasted and that toxic metabolites such as hydrogen peroxide are not produced. These goals are achieved by embedding redox-active Current Opinion in Biotechnology 2003, 14:590–596
centers deeply in an ‘insulating’ protein shell and by regulating electron transfer through sophisticated control mechanisms. Willner, Katz, Scheller and Schuhmann have summarized progress in using redox proteins for bioelectrocatalytical applications and have addressed design issues of electrode surfaces for efficiently relaying electrons to redox proteins in four excellent reviews [1,2,3,4]. In this review, we focus on protein engineering approaches for increasing electron transfer rates from electrode surfaces to redox proteins and discuss strategies for tailoring redox proteins to our needs in applications.
Electron transfer in redox proteins Electron transfer plays a pivotal role in biological functions essential to life, such as photosynthesis, respiration and metabolic pathways. In Marcus’ theory, three factors determine electron transfer within proteins [5]: reorganization energies (qualitatively reflecting the structural rigidity in the oxidized and reduced form); potential differences and orientations of involved redox-active sites; and the distances between redox-active sites and the intervening medium. The electrode and enzyme active site can be regarded as electron donor–acceptor pair. The protein or glycoprotein shell surrounding the active site becomes an effective kinetic barrier for heterogeneous electron transfer and can interfere with efficient direct electrical communication.
Recent advances in electrochemical applications of redox proteins Novel electrode surfaces that act as efficient interfaces for transferring electrons significantly broaden the repertoire of enzymatic reactions that are electrochemically accessible. Progress has been especially remarkable for P450 enzymes [6]. Recent highlights in biofuel cell research and chemical syntheses are summarized in Table 1. Additional innovations can be expected from fundamental research efforts, in which bioelectrochemistry is used as a tool for studying intermolecular biological electron transfer [7] and protein folding [8].
Challenges of bioelectrocatalysis employing redox proteins Being in its infancy, bioelectrocatalysis continues to be confronted with a broad range of challenges some of which are outlined below (Figure 1). Field conditions
Proteins exhibit high activities often at conditions close to their ‘natural’ environment. Glucose oxidase (GOx) for www.current-opinion.com
Protein engineering in bioelectrocatalysis Wong and Schwaneberg 591
Table 1 Bioelectrocatalytical highlights in biofuel cell research and chemical syntheses. Application
Examples
Novelty
Biofuel cell (anode/cathode)
Glucose oxidase/laccase Glucose oxidase/cytochrome c and cytochrome c oxidase Glucose oxidase/bilirubin oxidase Glucose oxidase/bilirubin oxidase Glucose dehydrogenase/bilirubin oxidase Glucose oxidase
High power output (0.78 V) [30] Switchable and tunable power output [31]
Interesting anodes
Glucose oxidase
Chemical syntheses (enzyme/application)
P450cam and myoglobin/styrene epoxidation P450cam/styrene epoxidation
instance, works well in a slightly acidic environment, but when employed in an implanted medical device its activity is compromised by the blood pH (7.3) and high chloride concentration (98–108 mM). Enzymatic properties Cofactor requirement
Cofactor regeneration poses a major challenge for many applications employing redox proteins. Cofactors such as NADH or NADPH are expensive and tend to be adsorbed at electrode surfaces. These adsorbed cofactors can subsequently be oxidized, leading to degradation of the cofactors and modifications of the electrode surfaces [9]. Enzyme instability
Maximum current output can often only be maintained for a short period of operation owing to ‘degradation’ of redox proteins and irreversible surface modifications [10]. Regarding aerobic cathodic systems, a major challenge is to run the reaction without reducing molecular oxygen to reactive species such as superoxide or peroxide that oxidatively damage the biocatalysts.
Power generation (2.4 mW, 0.52 V) with a grape [32] High power output (50 mW, 0.5 V) under physiological conditions [33] High power density (58 mW) under physiological conditions [34] A novel electrochemical communication. Gold nanoparticles act as an electron relay for the alignment of the enzyme on the conductive support and for the electrical wiring of its redox-active center [35] Increased electron diffusion coefficient by the tethering of redox centers to the backbone of the cross-linked redox polymer backbone through 13-atom spacer arms [36] Electrochemical-driven epoxidation of styrene with ultrathin polyion film [37] Styrene epoxidation in an electroenzymatic reactor [38]
provide linkages, and reduction of enzyme activity due to immobilization. Electron transfer mechanism in redox proteins
The electron transfer in redox proteins is tightly regulated to improve the fitness of the organism by avoiding waste of valuable energy resources and the generation of toxic intermediates. Regulation mechanisms within proteins are highly advanced (Figure 2) and well-gated on the molecular level by structural rearrangement within the proteins. For example, P450 BM-3 has a substrateinduced thermodynamic switch that regulates the electron transfer [12]. In the absence of substrate, no electron transfer from flavin mononucleotide (FMN) to heme occurs. This control mechanism prevents futile consumption of energy resources and avoiding the production of reactive and cell-damaging oxygen species such as hydrogen peroxide. Kinetic measurements for P450 BM-3 showed that the rearrangements within the protein for electron transfer, and not the oxygenation chemistry, are likely to be rate limiting. Conducting interface
Enzyme pretreatment
Oxidative pretreatments for cleaving the sugar moieties of redox proteins expressed in fungi and yeast are common approaches for boosting electron transfer rates by reducing the distance between redox proteins and electrode surfaces. However, strong oxidizing agents such as periodates often reduce enzymatic activity significantly [11]. Enzyme immobilization
Immobilization of redox proteins on electrode surfaces to achieve reproducible and fast electron transfer is often a trial and error process. Technical challenges include the even distribution of enzymes on electrode surfaces, the modification of enzymes or electrode surfaces to www.current-opinion.com
Electron transfer rates from redox proteins to electrode surfaces are key determinants of the overall performance and efficiency of bioelectrochemical devices. In the past few years, researchers have achieved remarkable progress in designing effective interfaces between redox proteins and conductive supports. Challenges encountered include electrode design, electrode surface functionalization for protein immobilization, irreversible adsorption and the degradation of cofactors, to name a few. Most approaches aim to optimize electron transfer rates by leaving the amino acid composition of the enzymes unchanged. Extensive research has been performed on mediated electron transfer [13], direct electron transfer between redox proteins and electrodes, electron transfer cascades via redox hydrogels, Current Opinion in Biotechnology 2003, 14:590–596
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Figure 1
Figure 2 Cytochrome P450 BM-3
Electrode NADPH
FAD
TC
FMN
Heme b
Fatty acid
Successful protein engineering strategies Nitric oxide synthase
Trimming the protein
NADPH
FAD
FMN
Heme b
L-Arg
Protein surface modification(s) TC
TC
Active-site mutation(s) L-Arg
Heme b
FMN
FAD
NADPH
Domain shuffling Cytochrome c oxidase Cyt c
CuA
Heme a
H+ Heme a /Cu 3 B
O2
Cytochrome cd1
e– Transfer Cyt c
Heme c
CG
Heme d1
NO2–
Current Opinion in Biotechnology
Field condition(s) (blood)
Four well-studied electron transfer chains in prominent redox proteins. Arrows indicate electron pathways and electron transfer is controlled by thermodynamics (TC, thermodynamic control), conformational gates (CG), and coupled electron/proton transfer (Hþ).
Enzyme properties (pH, temperature) Conducting interface design Understanding and tuning electron transfer within a redox protein
Challenges for protein engineering in bioelectrocatalysis Current Opinion in Biotechnology
Successes and challenges of protein engineering in bioelectrocatalysis.
and conducting polymer films [4]. Katz and coworkers [14] recently demonstrated an innovative approach for electrochemically driving oxidoreductases containing flavin adenine dinucleotide (FAD), or using NAD(P)þ. High bioelectrocatalytic activities were achieved especially with the GOx system, in which an apo-GOx (lacking its FAD) was reconstituted with an FAD cofactor that is covalently coupled to a phenylboronic-acid-functionalized gold electrode.
Protein engineering of redox proteins for electrochemical applications Protein engineering offers attractive solutions for overcoming physiological constraints. It improves enzymatic Current Opinion in Biotechnology 2003, 14:590–596
properties and enhances the electrochemical performance of bioelectronics by directing engineering focus on the redox protein, a key determinant in bioelectrocatalysis. Protein engineering technology has matured rapidly and many powerful genetic engineering tools are available [15]. General approaches in protein engineering include rational design, directed evolution (laboratory evolution), and combined efforts. Rational design
Rational design of proteins is driven by the hypotheses of researchers and computer models based on available crystal structures of the protein of interest. Site-directed mutagenesis is the method used in rational design for verifying deduced protein–function relationships. To rationally design redox proteins for biofuel cells and sensing applications several strategies have been used and are outlined below. Trimming the ‘fat’
The active site of an oxidoreductase is seldom found on the protein surface. By removing the amino acids that are not essential for maintaining protein function and structure, one can open up the active site and position a conducting support in close proximity to it. Two prominent examples in which this strategy was successfully used for improving electron transfer rates are microperoxidases and laccase from Trametes versicolor (LCCI). www.current-opinion.com
Protein engineering in bioelectrocatalysis Wong and Schwaneberg 593
Microperoxidase-11 is an undecapeptide (N-Val-GlnLys-Cys-Ala-Gln-Cys-His-Thr-Val-Glu) with a heme c group attached through thioester bonds at the two cysteine residues. It was obtained by enzymatic digestion of cytochrome c, and still exhibits electrochemical properties [16]. Microperoxidase-11 reveals several advantages over peroxidases in peroxide reduction: it is much smaller in size, exhibits high stability and direct electrical communication with many different electrodes because of its heme exposure, and has been successfully employed in biofuel cells. With laccase, Palmore and co-workers [17] expressed a truncated laccase LCCI in Pichia pastoris as the heterologous host. The amino acid sequence of this truncated laccase is identical to that of LCCI except that the final 11 amino acids (PIYDGLSEANQ; in single-letter amino acid code) at the C terminus of LCCI are replaced by a single cysteine residue. On the basis of their analysis of available crystal structures of laccase (Protein Data Bank [PDB] code 1a65 from Coprinus cinereus) and ascorbate oxidase (PDB code 1AOZ, a similarly structured copper oxidase from Cucurbia pepo medullosa), they hypothesized that the rate of heterogeneous electron transfer might increase if they widened access to the type 1 copper site of LCCI. This hypothesis was investigated by reducing the number of amino acid residues between the last histidine that binds the type 1 copper ion in the active site of LCCI (His478) and the C terminus (Gln519). Introduction of a cysteine residue at the C terminus of laccase provides a chemical target (i.e. thiol) for selective modification. Results from electrochemical studies showed that the redox potential of the active site of truncated laccase was shifted to more negative values than those found in other fungal laccases. Electron transfer from the electrode surface to the protein was observed for the truncated LCCI in contrast to no cyclic voltammogram recorded for the native enzyme.
ferrocenecarboxylic acid, to GOx for improving sensitivity and stability of glucose biosensors. The modified GOx showed comparable Km and kcat values to those of the wild-type enzyme; however, in the presence of ferrocenecarboxylic acid the polylysine GOx retained 90% of its activity, whereas native GOxs retained only 22% [20]. Halliwell and coworkers [21] introduced several tags in a L-lactate dehydrogenase (N- and C-terminal (poly)histidine tags and a C-terminal cysteine) enhancing the current density at the bioanode by a factor of 5 to 7 at certain lactate concentrations.
Protein surface modifications
Directed evolution
Protein surface modifications to achieve better heterogeneous electron transfer and cell performance have recently been reported. Gorton and co-workers studied the effect of His-tag and surface-exposed cysteine residues on the electrochemical properties of horseradish peroxidase (HRP). Non-glycosylated HRP containing a six-histidine tag at the C terminus, C(His)rHRP, was adsorbed on a pre-oxidized gold electrode and generated a more than 30-fold increase in electron transfer rate [18]. Introducing additional cysteine residues, preferably at the C terminus, led to a favorable direct proton-coupled or proton-gated electron transfer. The improved electron transfer rates were explained on the molecular level by surface interactions that play a dominant role in immobilizing and orienting HRP on electrode surfaces [19]. Another successful example was GOx, where a polylysine chain was added at the C terminus. The polylysine chain was added to anchor the electron transfer mediator,
The lack of a profound understanding of the relationship between protein structure and electron transfer hinders scientists from rationally designing efficient interfaces and pathways for transferring electrons (Figure 1). Directed evolution does not require knowledge of structures or functions and allows the tailoring of enzymes to our needs by mimicking Darwinian evolution [26]. Directed evolution will be very valuable in discovering the fundamental design principles of electron transfer in redox proteins when applied to proteins of known structure. In a directed evolution experiment, a pool of mutated enzymes is created via modification of the DNA and resulting proteins with beneficial properties are identified by selection or screening. This iterative process is repeated until the desired trait is improved. Random mutagenesis by error-prone polymerase chain reaction (epPCR) is most commonly used for creating diversity on the genetic level. The mismatches are achieved by
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Active-site mutations
Introducing mutations in the active site is an additional option for improving intermolecular electron transfer and substrate specificity, as shown by Davidson who studied the mechanisms of catalysis and electron transfer in methylamine dehydrogenase [22]. In this study, Phe55 of the a subunit, located where the substrate channel from the enzyme surface opens into the active site, was substituted with alanine. The engineered methylamine dehydrogenase (Phe55Ala) improved the sensitivity of a histamine sensor up to fourfold owing to the lower Km value [23]. Domain shuffling
A new enzyme engineering concept was introduced by Gilardi’s group who rationally constructed P450s that displayed improved electrochemical properties by assembling catalytic and electron transfer modules using ‘gene fusion’ techniques [24]. Domain shuffling is a widely used genetic engineering technique for swapping functional modules among closely related proteins. In addition, Gilardi’s group engineered a nucleic acid binding protein, rop, into a heme-binding protein that displays typical spectrophotometric and electrochemical properties of a heme protein [25].
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Table 2 Comparison of rational design and directed evolution. Aspect
Rational design
Directed evolution
Methods for mutating DNA Understanding of structure–function relationships Availability of crystal structure Key to success Experimental time for mutagenesis
Site-directed mutagenesis Important prerequisite
epPCR and recombination methods Not important
Important prerequisite A reliable model Short: single or few site-directed mutagenesis experiments 1–2 mutations
Not important A reliable high-throughput screening method Long: developing of a robust screening protocol depending on library size and iterative cycles With epPCR often 1–3 mutations
Determined by experimenters Determined by experimenters
Random Biased by employed polymerase and error-prone conditions
Number of mutations per mutagenesis experiment per gene Location of the mutation(s) Nucleotide substitution
employing DNA polymerase with low fidelity. The fidelity can further be reduced by addition of Mn2þ and/or an imbalanced nucleotide concentration [27]. Combining diverse genes that were selected for function by natural evolution can be an alternative method to epPCR for generating genetic diversity. Many powerful recombination methods, such as StEP, DNA shuffling, nonhomologous random recombination [28], SHIPREC, ITCHY and RACHITT, have been developed for molecular breeding [15]. Proteins will encounter operating conditions in electrochemical applications that strongly deviate from the physiological prerequisites (e.g. controlled electron flow, isolating protein shell) that natural selection imposes. For instance, heterogeneous electron transfer from redox proteins to electrodes is a property that has not been under selective pressure in nature. Accumulating new mutations by random mutagenesis may be more promising in ‘evolving’ this novel function than recombining proteins designed by nature that do not have the ‘trait’. However, DNA shuffling methods that introduce random mutations might be valuable alternatives. In a directed evolution experiment, a rapid screen or selection that reflects desired functions is the key to success. Enzymatic properties that are important for bioelectrocatalytical applications include reduced Km values for improving biosensors, increased tolerance towards field conditions (e.g. temperature, pH, inhibitors) and higher catalytic activities for boosting the power output of biofuel cells. Directed evolution has been proven a powerful tool for evolving all of these properties. An important lesson from directed evolution is that it has been especially easy to evolve traits that have never been under selective pressure in nature. Hence, it is encouraging to evolve heterogeneous electron transfer by directed evolution even though, at least not to our knowledge, it has not been reported yet. In our opinion, colorimetric or fluorometric assays based on electron transfer mediators that show color change upon Current Opinion in Biotechnology 2003, 14:590–596
a switch of oxidation state seem to be promising systems for directed evolution. A more costly, but also feasible, approach is to use potentiostats in a high-throughput format. Designing a protein engineering experiment requires weighing the pros and cons of rational design and directed evolution (Table 2) and needs the experimental constraints of the screening or selection system to be taken into consideration. Combining rational design and directed evolution (semi-rational design or rational evolution) often substantially accelerates the progress in protein engineering [29].
Conclusions Researchers are now borrowing a page from biology’s manual to create tailor-made proteins for applications in sensors, biofuel cells and syntheses of pharmaceuticals and fine chemicals. Despite the far-stretching potential in applications, there are considerable challenges in discovering fundamental structure–function relationships before we can understand and tune electron transfer. Rational design has proven its potential and many success stories in directed enzyme evolution have shown that it is especially easy to improve traits never required for biological function, such as stability or activity in a nonnatural environment or activity towards a non-natural substrate. This raises the hope that redox proteins can be engineered successfully, paving the way to a biotechnological revolution in biosensors, biofuel cells and biocatalysis.
Update Recent investigation showed that trimethylamine dehydrogenase mutant (Tyr422Cys) could be successfully immobilized on electrode surface that was chemically modified with redox polymer, enabling direct electrical communication between the enzyme and electrode [39]. Amperometric bi-enzyme sensor using NADH regeneration was also recently reported and this solves the fundamental difficulty in cofactor recycling [40]. www.current-opinion.com
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Acknowledgements We thank the Office of Naval Research (ONR) for support and Kang Lan Tee for checking the manuscript.
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impressive electron transfer rate of the reconstituted bioelectrocatalyst is 5000 s1, compared with 700 s1 with the natural cosubstrate oxygen.
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37. Munge B, Estavillo C, Schenkman JB, Rusling JF: Optimization of electrochemical and peroxide-driven oxidation of styrene with ultrathin polyion films containing cytochrome P450(cam) and myoglobin. Chembiochem 2003, 4:82-89. 38. Mayhew MP, Reipa V, Holden MJ, Vilker VL: Improving the cytochrome P450 enzyme system for electrode-driven biocatalysis of styrene epoxidation. Biotechnol Prog 2000, 16:610-616. 39. Loechel C, Basran A, Basran J, Scrutton NS, Hall EAH: Using trimethylamine dehydrogenase in an enzyme linked amperometric electrode—Part 2: Rational design engineering of a ‘wired’ mutant. Analyst 2003, 128:889-898. 40. Mak KKW, Wollenberger U, Scheller FW, Renneberg R: An amperometric bi-enzyme sensor for determination of formate using cofactor regeneration. Biosens Bioelectron 2003, 18:1095-1100.
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