Semisynthetic and Biomolecular Hydrogen Evolution Catalysts

Dec 15, 2015 - In this Forum Article, progress in engineering proteins to catalyze H2 evolution from water is discussed. The artificial enzymes descri...
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Semisynthetic and Biomolecular Hydrogen Evolution Catalysts Banu Kandemir, Saikat Chakraborty, Yixing Guo, and Kara L. Bren* Department of Chemistry, University of Rochester, Rochester New York 14627-0216, United States S Supporting Information *

ABSTRACT: There has been great interest in the development of stable, inexpensive, efficient catalysts capable of reducing aqueous protons to hydrogen (H2), an alternative to fossil fuels. While synthetic H2 evolution catalysts have been in development for decades, recently there has been great progress in engineering biomolecular catalysts and assemblies of synthetic catalysts and biomolecules. In this Forum Article, progress in engineering proteins to catalyze H2 evolution from water is discussed. The artificial enzymes described include assemblies of synthetic catalysts and photosynthetic proteins, proteins with cofactors replaced with synthetic catalysts, and derivatives of electron-transfer proteins. In addition, a new catalyst consisting of a thermophilic cobalt-substituted cytochrome c is reported. As an electrocatalyst, the cobalt cytochrome shows nearly quantitative Faradaic efficiency and excellent longevity with a turnover number of >270000.

1. INTRODUCTION AND SCOPE A key challenge facing society is the development of renewable energy resources that have a modest environmental footprint.1,2 Solar energy is attractive for being abundant and renewable, but its wide implementation requires the development of new storage technologies. One approach is to perform artificial photosynthesis, in which a fuel-forming chemical reaction is driven by light. In this area, a major focus has been on photochemical water splitting to form an energy-dense and clean-burning fuel, hydrogen (H2):3−9 H 2O → H 2 +

1 O2 2

Figure 1. Generalized catalytic cycle for a H2 evolution catalyst forming H2 through a heterolytic mechanism in which a metal hydride is protonated.

When the reductive side of water splitting to produce H2 is performed, a source of electrons is needed. One approach is to supply the electrons electrochemically at a potential that is sufficient to achieve H2 evolution at a reasonable rate given the reaction conditions and catalyst overpotential. The other approach is to use a photosensitizer to provide electrons through photoinduced charge transfer (Figure 2). Each approach offers unique opportunities and challenges. In

(1)

A successful solar water-splitting device must achieve high-yield photoinduced charge separation and catalysis of water oxidation and reduction according to the two half-reactions: H 2O →

1 O2 + 2e− + 2H+ 2

2H+ + 2e− → H 2

(2) (3)

Furthermore, a membrane is needed to separate the products from each other while also allowing transport of electrons and protons, and the conditions for the oxidation and reduction reactions must be compatible with this membrane.2,8 The challenges of integrating these requirements have led many researchers to focus on investigating water oxidation (eq 2) or proton reduction (eq 3) individually. Here, we will focus on the fuel-forming proton reduction reaction, which may be driven electrochemically or photochemically. A generalized catalytic cycle is shown in Figure 1. © XXXX American Chemical Society

Figure 2. Schematic of a photocatalytic system for H2 generation using a photosensitizer (red star) and a sacrificial electron donor (D/D+). Special Issue: Small Molecule Activation: From Biological Principles to Energy Applications Part 3 Received: September 4, 2015

A

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into H2, for performing the reverse reaction, and for transporting the reactants and products. While inorganic and bioinorganic chemists have done well in mimicking the core activity of hydrogenases, including introducing proton-transfer sites at an active site,21−29 the field is a long way from understanding and reproducing the many coordinated actions of these enzymes. The use of protein catalysts is of high interest because this approach may provide a pathway to producing engineered enymes that incorporate mechanisms for moving substrates and products in addition to making H2. The use of nature’s hydrogenase enzymes in assemblies for light-driven H2 evolution is a promising complementary approach30−40 but is outside of the scope of this Forum Article. Furthermore, we will limit our discussion to systems using catalysts composed only of earth-abundant elements.

electrocatalysis, electrons are delivered to the catalyst via an electrode at a known potential. By variation of the experimental conditions, it is possible to extract information on the reaction rate, overpotential, and mechanism.10,11 A complication of electrocatalysis is that it occurs at a liquid−solid interface, which introduces a reaction microenvironment that differs from the bulk solution and from conditions under which the catalyst is typically characterized. Furthermore, the electrode material must be compatible with the catalyst, and there is the possibility of catalysis occurring through both homogeneous and heterogeneous mechanisms.12 In contrast, the use of a photosensitizer allows the reaction to be carried out in solution and without interaction with a surface. However, success with a photocatalytic system requires that the photosensitizer be compatible with the catalyst. It also may require an electron relay, and a sacrificial electron donor also is needed. Overall, photocatalysis is significantly more complex than electrocatalysis. It requires photoinduced charge-transfer steps to deliver two electrons to the active site, and two protons also must be supplied. These steps must be coordinated properly for efficient H2 evolution.13,14 Because photocatalysis requires these multiple components to function properly, the number of turnovers for a catalyst in a photocatalytic system is typically much lower compared to its activity as an electrocatalyst. This Forum Article will focus on the use of engineered biomolecular catalysts and biomolecular scaffolds supporting synthetic catalysts for H2 production from water. The development of biomolecules for H2 production is inspired by the enviable properties of hydrogenases. Hydrogenases are metalloenzymes that catalyze the reversible reduction of protons to H2. They operate in water under mild conditions at a very low overpotential,15 meaning that they catalyze the reaction at a potential close to the thermodynamic potential. Some hydrogenases are extremely fast, with turnover frequencies (TOFs) for H2 production approaching 10000 mol of H2 (mol of catalyst)−1 s−1.15 The major classification of hydrogenases is based on their active sites: mononuclear iron [Fe], dinuclear iron [FeFe], or iron−nickel [NiFe] (the binuclear sites are shown in Figure 3). While the active site is

2. BIOMOLECULAR AND MACROMOLECULAR ASSEMBLIES WITH SYNTHETIC CATALYSTS An approach to taking advantage of the attractive features of biomolecular structures to create H2 evolution catalysts is to assemble a synthetic catalyst with a biomolecule. The biomolecule may have a binding pocket to accommodate the catalyst and provide it with a specific desirable microenvironment, or it may have in place machinery for long-range photoinduced electron transfer to facilitate electron delivery and prevent energy-wasting recombination reactions. This strategy also confers water solubility on the synthetic catalyst, most of which do not function in 100% water. Here, we describe examples of complexes between synthetic molecular H2 evolution catalysts and macromolecules prepared with these goals in mind. Most of the systems described are activated photochemically, and their performance is summarized in Table S1. 2.1. Complexes of Catalysts with Photosystem I (PSI). Photosystem I is a chlorophyll-binding photosynthetic protein complex that performs photoinduced charge transfer across the thylakoid membrane with high thermodynamic efficiency.14,41,42 The primary electron donor, P700, transfers electrons through an electron-transfer chain consisting of redox cofactors, terminating with the FB iron−sulfur cluster with a midpoint potential of −580 mV vs SHE (Figure 4). FB transfers electrons to ferredoxin−NADP+ reductase via ferredoxin, culminating with the reduction of NADP+ to produce the biological energy carrier NADPH. Electrons are supplied to PSI by soluble electron-transfer proteins (plastocyanin or cytochrome c6). PSI is an attractive module for converting light energy to a charge-separated state, which then may be used to do chemistry such as proton reduction. To put PSI to use for photochemical H2 production from water, PSI was modified with Co(dmgH)2pyCl (dmgH2 = dimethylglyoxime; py = pyridine), a well-studied protonreducing catalyst in the cobaloxime family (structure 1 in Figure 5; the strategy is illustrated in Figure 4 with a different catalyst).43 A turnover number (TON) of 5200 mol of H2/mol of PSI and a TOF of 170 mol of H2 (mol of PS1)−1 min−1 were reported upon illumination for 1.5 h in the presence of sodium ascorbate as a sacrificial electron donor and cytochrome c6 as an electron mediator. This TON compares well to values for cobaloxime catalysts paired with synthetic photosensitizers, which give TON values of up to 9000 with respect to the photosensitizer.44,45 Unlike many synthetic systems, the PSI− Co system performs in 100% water and uses no heavy metals such as ruthenium- or platinum-based photosensitizers.

Figure 3. Structures of the active sites of (A) [FeFe] and (B) [NiFe] hydrogenases. In part B, X is an exogenous ligand such as H− or OH−.

where the bond making and bond breaking occurs, other components of the polypeptide structure also are important for hydrogenase activity. The protein matrix of hydrogenases provides a defined pocket for the cofactor and ligands for active-site metal ions.15 The active site is linked to the protein surface by pathways for the transfer of reactants and products. Chains of redox cofactors deliver electrons to or from electrontransfer partners at a potential and rate appropriate for the reaction. Ionizable amino acids and/or buried water molecules facilitate the movement of protons to and from the catalytic site,16,17 and gas-transfer channels transport H2 between the buried active site and the surface.18−20 Hydrogenases are finely tuned machines for assembling two electrons and two protons B

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Figure 4. (A) Schematic for photocatalytic H2 production from a PSI−molecular catalyst hybrid. The catalyst location is not known. (B) Schematic of the PSI cofactors and proposed location of the catalyst. A nickel catalyst is shown, but a similar scheme is used for activation of a cobaloxime catalyst. Reprinted with permission from ref 50. Copyright 2013 American Chemical Society.

studies of the loss of activity may provide hints on how to develop a system with the same high photocatalytic rates but with greater longevity. Some nickel catalysts that contain built-in proton shuttles are known for having high electrocatalytic activity and low overpotentials.47,48 One of these catalysts, ([Ni(P2PhN2Ph)2](BF4)2 (2); Figure 5) was attached to PSI to yield a photocatalytic assembly, Ni-PSI. In the presence of ascorbic acid and cytochrome c6, this assembly produces H2 upon illumination in fully aqueous conditions for about 3 h, with a TON of 1870 mol of H2 (mol of PSI)−1. To compare to a fully synthetic system, in the presence of an organic dye photosensitizer, this same catalyst in an acidic water (pH 2)/ acetonitrile mixture gave 2700 turnovers relative to the catalyst over 150 h, with activity limited by photosensitizer decomposition.49 The TON of the Ni-PSI complex is somewhat lower than the TON for the synthetic multicomponent system in solution, but Ni-PSI produces H2 at a much higher rate and under mild aqueous conditions.50 Furthermore, compared to the synthetic photosensitizer−nickel multicomponent system,49 the rates of H2 production are about 10 times greater on a per nickel basis for Ni-PSI. In contrast to this finding, many synthetic systems in which photosensitizers have been physically linked to catalysts have underperformed relative to bimolecular systems.51,52 The nickel catalyst is attached to PSI through self-assembly. An alternative approach is to engineer a nickel-catalyst-carrying protein to deliver the catalyst to the FB site on PSI. PSI has a flavodoxin electron-transfer partner, and the researchers inserted the nickel catalyst into the flavin binding site in the apoflavodoxin; the resulting Ni-apoFld was then allowed to dock with PSI. With a 30-fold excess of Ni-apoFld combined with PSI, H2 production rates determined with respect to PSI were 2-fold higher than those for PSI-Ni. H2 production leveled off after 3 h and gave a TON of 2825 mol of H2 (mol of PSI)−1. The use of protein−protein interactions to deliver a catalyst to an electron donor is a clever strategy that could be applied to other protein-based catalysts for H2 production. Furthermore, there is a wealth of biophysical data on protein−protein interactions, including Kd values, on/off rates, and interaction sites53,54 that can be drawn upon to design protein-based

Figure 5. Structures of selected catalysts used in the production of biohybrid systems for H2 evolution.

Furthermore, PSI−cobaloxime yields better results in terms of the TON than some integrated synthetic systems for photocatalytic H2 production in which photosensitizers and cobaloxime catalysts are linked.44 PSI−cobaloxime gives a high TON for a photocatalytic system. Additional studies would help the community to understand what factors contribute to its high activity, which could yield even better performing systems. One intriguing question that the authors note regards a mismatch of the reduction potentials of the FB electron donor (−580 mV vs SHE) and the CoII/I potential of Co(dmgH)2pyCl (−880 to −740 mV). Furthermore, cobaloxime CoII/I potentials have been shown to shift to more negative values by ∼100 mV when bound in the hydrophobic interior of apo-Mb,46 and such a shift is possible for this system because the catalyst is expected to bind PSI through hydrophobic interactions. Given the excellent performance of this system, fast transfer of the first electron to CoII to initiate the catalytic cycle (Figure 1) is expected, and it would be very interesting to learn more about this process. A factor limiting the assembly’s performance is that the longevity is up to 1.5 h. The authors note that the catalyst dissociates from PSI during photocatalysis, but whether it is degraded first or released intact is not clear.14 Additional C

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incorporation of a catalyst into a specific site within a protein provides a route to understanding how the protein matrix influences the catalyst activity and may facilitate the introduction of defined electron- and proton-transfer pathways. In one example of this approach, a diiron complex mimicking [FeFe] hydrogenase was introduced into the protein nitrobindin. Nitrobindin is a β-barrel protein that normally binds a heme within its large internal cavity. In place of heme, a synthetic [FeFe] mimic was incorporated by covalent attachment via a maleimide moiety on the catalyst to an introduced Cys residue (Figure 6). Compared to the [FeFe] mimics

donor−acceptor systems for catalysis and also to better understand the mechanism. 2.2. Peptide−Catalyst Assemblies. The PSI−catalyst systems are a “top-down” approach to developing biohybrids that catalyze solar H2 production. A “bottom-up” approach is to build an active site into a synthetic biopolymer. Thus, far, these systems have consisted of synthetic catalysts bound to peptides.55 A popular target in this area is inspired by the structures of nature’s hydrogenases, and the [FeFe] hydrogenase active site has received the most attention (Figure 3A). This work was pioneered in 2007 by the demonstration that a diiron carbonyl complex can be bound to a CXXC motif within a helical peptide to build a mimic of the [FeFe] site.56 The attachment of the diiron mimic to a peptide provides water solubility and, potentially, second-sphere interactions. Since that time, related constructs have been shown to have catalytic activity for H2 production either as an electrocatalyst or in a photocatalytic system. In one example, a diiron cluster (structure 3, Figure 5) was attached to a synthetic peptide through an unnatural dithiol amino acid.57 The construct has some α-helical content, as seen by circular dichroism (CD) spectroscopy. H2 was produced upon illumination using [Ru(bpy)3]2+ as a photosensitizer and ascorbic acid as an electron donor to yield a TON of 84 over 2.3 h. Electrochemically, an irreversible reduction wave at −1.1 V vs SHE was observed by cyclic voltammetry (CV) at pH values ranging from 3.6 to 5.5. The catalytic current has an unusual and interesting pH dependence, with an abrupt decrease in the peak current between pH 4.0 and 4.5. It would be interesting to learn whether this dependence is seen only in the electrocatalysis experiments or also when paired with a photosensitizer. In a related strategy, diiron clusters were attached to peptides by iron coordination to sulfur atoms of Cys residues in a CXXC motif.58,59 These systems were constructed either from a synthetic 18-mer peptide58 or a 104-amino acid apocytochrome c.59 The CD spectrum of the [FeFe] apocytochrome reveals a minimal secondary structure. Both complexes were active in photocatalytic systems with [Ru(bpy)3]2+ or a derivative as the photosensitizer, giving 82 (for the 104-mer) and 9 (for the 18mer) turnovers. For the smaller peptide complex, the chromophore was attached to the same peptide as the catalytic cluster to give a unimolecular system. Surprisingly, no activity was observed with bimolecular activation of the peptide− cluster complex with [Ru(bpy)3]2+. In contrast, the 104-mer [FeFe] peptide can be activated by [Ru(bpy)3]2+ in a multicomponent system. These systems represent the initial steps toward building designer enzymes that mimic both the active-site structure and activity of [FeFe] hydrogenases. An important difference in the active site of these artificial hydrogenases compared to [FeFe] hydrogenase is that the active site in the native enzyme includes a built-in amine proton shuttle (Figure 3A), and it has been shown that assembling [FeFe] hydrogenase with an altered cluster lacking the amine yields an inactive enzyme.60 The incorporation of this feature into these synthetic hydrogenases is a refinement worth considering if it is technically feasible. 2.3. Other Protein−Catalyst Hybrids. Biohybrid catalysts prepared by assembling synthetic catalysts with small, wellcharacterized proteins will be the focus of this section. This approach to preparing artificial hydrogenases has the advantage of utilizing biomolecules that are structurally well-defined and that can be engineered, manipulated, and characterized in detail using kinetic, structural, and biophysical methods. The

Figure 6. Introduction of a diiron active site into nitrobindin through covalent attachment. Reprinted with permission from ref 61. Copyright 2014 American Chemical Society.

attached to peptides, higher activity (TON of 130) for H2 evolution induced with a photosensitizer was achieved.61 This system is appealing because it makes use of a structurally welldefined protein to host the catalyst. As a result, there is the possibility of introducing mutations in the nitrobindin host to alter its interactions with the catalyst and possibly enhance the activity or stability. This system also is distinguished by making use of a stable covalent bond for attaching the catalyst to the polypeptide, which contrasts with the weaker coordinate bonds used in the [FeFe] mimic−peptide systems and the hydrophobic interactions used to attach catalysts to PSI. Another example of the introduction of a synthetic catalyst into a protein cavity is the insertion of cobaloxime derivatives Co(dmgH)2 and Co(dmgBF2)2 (structure 4, Figure 5) into the heme pocket of apo-sperm whale myoglobin (apo-Mb).46 The planar catalysts fit naturally into the heme pocket, and the protein provides an axial histidine imidazole to bind the cobalt; modeling studies suggest that it is the proximal His93. Electrocatalysis studies reveal that the cobaloxime−Mb complex evolves H2 in neutral-pH water at a low overpotential (200 mV). The catalyst also was activated by chemical reduction and by use of photosensitizers and gives up to 10 turnovers. The authors proposed that the catalyst is deactivated via a catalytic intermediate such as a CoIII-H species (Figure 1), which may transfer a hydride to the glyoximato ligand. This transfer could take place if the deactivation step is fast relative to the delivery of the second electron to yield CoII-H, which then evolves H2 upon protonation. Alternatively, it is possible that the electron-transfer steps are sufficiently fast, but protonation of CoII-H, which will be a more potent hydridetransfer species, is slow. It would be interesting to learn D

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Figure 7. (A) CoMP11-Ac and (B) Ht c-552 (PDF: 1YNR).75

approach is to engineer an existing biomolecular structure to introduce hydrogenase activity by metal substitution and/or mutation. This approach allows the use of soluble, structurally characterized, and robust biomolecules in catalyst design and engineering. These engineered biomolecular catalysts are more likely than those prepared as synthetic catalyst−protein hybrids to have a well-defined structure at the active site, facilitating characterization, mechanistic study, and active-site engineering. The first demonstration of hydrogenase activity in a modified non-hydrogenase protein was the 1988 report of H2 evolution catalyzed by nickel-substituted rubredoxins from three different Desulfovibrio species.65 Rubredoxins are electron-transfer proteins with a single iron coordinated by four cysteine thiolate side chains in a pseudotetrahedral site. Substitution of iron with nickel creates a nickel rubredoxin (NiRd) with an active site that resembles the nickel site in [NiFe] hydrogenases (Figure 3B). Indeed, in [NiFe] hydrogenases, the nickel is the redoxactive ion and thus the site where proton reduction and H2 oxidation are expected to occur.15 A recent report has significantly extended the studies of NiRd by investigating its activity as an electrocatalyst and also in the presence of a photosensitizer.66 From the electrochemical studies, the authors measure an overpotential of 540 mV, which is high but in line with and even lower than values seen for many synthetic catalysts.6 The authors also identified a proton-coupled electron-transfer (PCET) process similar to that seen for [NiFe] hydrogenases and found that the initial reduction step is rate-limiting. With some analysis of the active-site structure and mutants, it is hoped that the authors can identify the group that participates in the PCET process. In addition, the effects of the nickel reduction potential on the reaction would be interesting to learn. There is an extensive literature on the effects of the metalloprotein active site structure on the redox potential; this area is particularly well developed in iron−sulfur proteins, for which hydrogen bonding to Cys ligands plays a dramatic role in tuning the potential.67 Metal substitution also was utilized to introduce hydrogenase activity into Mb. In this case, the native iron protoporphyrin IX was replaced with cobalt protoporphyrin IX (CoP), yielding Co-Mb.68 CoP alone is an electrocatalyst for proton reduction in acetonitrile (solubility in water is minimal), while CoP incorporated into the globin evolves H2 from water near neutral pH. Photocatalytic H2 production activity of Co-Mb using a [Ru(bpy)3]2+ photosensitizer showed a 4-fold increase (to a TON of 520) relative to CoP in solution. Surprisingly, a decrease in the activity of Co-Mb is seen below neutral pH.

whether the engineering of more efficient electron- and/or proton-transfer pathways to the active site increases the longevity by preventing this deactivation. Indeed, Mb evolved to bind O2 reversibly, which requires insulation of the active site from protons and electrons, and it may not provide efficient electron-transfer pathways in comparison with nature’s electron-transfer proteins or redox enzymes that are evolved to conduct electrons.62 However, the Mb protein scaffold was shown to support higher H2 evolution activity by another strategy, as discussed below. In a related approach to making use of a protein scaffold to support photocatalytic H2 production, an electron-transfer protein (ferredoxin) was used to provide an electron relay between an attached [Ru(bpy)3]2+ donor and a Co(dmgBF2)2· 2H2O (structure 4, Figure 5) acceptor.63 The hybrid photochemically evolves H2 from water with a TON of 210 ± 60 with respect to the photosensitizer. An attractive aspect of this system is that ferredoxin is a small, easily manipulated, structurally well-defined protein that facilitates mechanistic and biophysical studies. Indeed, the researchers employed transient absorption spectroscopy to show that electrons are transferred from a RuII* excited state to reduce the catalyst to CoI. They also showed that the presence of the [2Fe−2S] cluster in the ferredoxin is necessary for activity, indicating that it is an electron-transfer site. A number of intriguing questions remain to be addressed in this system. In particular, characterizing the elementary electron-transfer steps and understanding the role of the iron−sulfur cluster in electron transfer in the context of Marcus theory would be enlightening. For example, the authors note that the driving force for electron transfer from the [2Fe−2S] cluster to the Co(dmgBF2)2 complex is low, which would yield a rapid electron-transfer rate only if the reorganization energy is also low. Interestingly, this is similar to many electron transfers in biology that occur at low driving force and with low reorganization energy.64 Additional investigations of the electron-transfer mechanism in this system would provide guidance on the design of variants with enhanced activity and also contribute to our fundamental understanding of photocatalytic H2 production by biological molecules.

3. ENGINEERED BIOMOLECULAR CATALYSTS Incorporating a synthetic catalyst into a protein or other macromolecule in a manner that creates a desired active-site structure is challenging because the biomolecule did not evolve to bind or activate the synthetic catalyst. A complementary E

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that of CoMP11-Ac but with the porphyrin contained within a heme pocket. The heme pocket then may be manipulated to alter the active site environment,78−80 and the polypeptide matrix has the potential to enhance the catalyst stability.68 We chose the heme protein Hydrogenobacter thermophilus cytochrome c552 (Ht c-552) in this work. Like MP-11, Ht c-552 contains a c-heme group that is covalently attached to the polypeptide via two thioether bonds in a CXXCH motif, with the His binding on the proximal side of the heme iron. The distal heme ligand is the Met61 thioether side chain (Figure 7B), which was mutated to Ala to open up a ligand-binding site at the heme.81−83 Ht c-552 is an attractive scaffold to use in this work because it is small (80 amino acids), highly water-soluble, and highly stable, even at temperatures as high as 100 °C.79,84 Here, we demonstrate that the cobalt-substituted M61A mutant of Ht c-552 (Ht-CoM61A) is an electrocatalyst for H2 evolution from water capable of achieving very high TON values. 4.1. Materials and Methods. Sample Preparation. HtM61A was expressed and purified using methods described in detail elsewhere.85 Water used in all steps was doubly deionized and had a resistivity of >18 MΩ. To demetallate the heme, the purified protein sample (20−30 mg) was exchanged to 20 mM NH4OAc, lyophilized, and dissolved in 3 mL of glacial acetic acid with 5 drops of concentrated hydrochloric acid in a Schlenk flask. Oxygen was removed by three to five freeze− pump−thaw cycles. The heme iron was reduced with 80 mg of FeCl2 added under argon.86 Starting from this point, the demetalation and metal-insertion steps were performed with minimal exposure to light. The solution was gently stirred for 30 min until a bright-purple color characteristic of the demetalated porphyrin was observed, when the acid was removed under vacuum. To ensure thorough removal of acid, the product was rinsed with 3 mL of doubly deionized water, which was then removed under vacuum. To chelate free iron, the sample was dissolved in a 50 mM sodium acetate buffer with 100 mM ethylenediaminetetraacetic acid, pH 5.5. A PD-10 column (GE Life Sciences) was utilized to exchange the sample to 50 mM NaOAc, pH 5.5. Demetalated protein was quantified by absorption spectroscopy using an extinction coefficient of 81 mM−1 cm−1 at the Soret band maximum, 396 nm.87 Typical demetalated cytochrome yields were 20−30%. A 500-fold molar excess of Co(OAc)2 relative to porphyrin was dissolved in 50 mM NaOAc, pH 5.5, and heated at 75 °C in a bomb flask for 1 h under argon. A color change to brownish red, indicative of cobalt insertion, was observed. The resulting cobalt derivative of Ht-M61A (Ht-CoM61A) was exchanged to 50 mM sodium phosphate, pH 7.0, using an Amicon stirred cell concentrator fitted with a 3 kDa molecular weight cutoff membrane. Purification of Ht-CoM61A was as described elsewhere for Ht-M61A.85 The typical yield of cobaltsubstituted protein relative to the starting material was 10%. To prepare Ht-ZnM61A for control experiments, a 100-fold molar excess of Zn(OAc)2 relative to demetalated protein (HtPM61A) was dissolved in a 50 mM NaOAc buffer, pH 5.5. Subsequent steps were as described elsewhere.88 Protein Characterization. UV−vis absorption spectra were measured on a Shimadzu UV-2401PC spectrophotometer using a 1-cm-path-length quartz cell. Ht-CoIIIM61A was quantified using a molar extinction coefficient of 161 mM−1 cm−1 at 421 nm.70,89 Reduction of Ht-CoIIIM61A (form as isolated) to HtCoIIM61A was carried out under nitrogen with a 500-fold excess of Na2S2O4. CD spectra were collected on a Chirascan Plus spectropolarimeter at ambient temperature (298 K). For

This effect of the pH was attributed to protonation of histidines within the active site (His64 and His97). To test this hypothesis, Co-Mb mutants H64A, H97A, and H64/97A were prepared. The H64A and H64/97A mutants were found to have increased activity at pH 6.5 relative to Co-Mb, whereas H97A had decreased activity. The lower activity of H97A may be a result of higher CoP lability. This result illustrates that mutation can modulate the activity of an engineered protein with H2 evolution activity. In a related study, the four-helix bundle heme protein cytochrome b562 was reconstituted with CoP.69 Cytochrome b562 has His/Met heme axial ligands, and Met was mutated to Ala, Asp, and Glu to potentially introduce a proton relay. The cobalt-substituted cytochrome was found to catalyze proton reduction in the presence of a photosensitizer and a sacrificial electron donor, with TON values of 120−310 reported over 7 h. This cobalt−porphyrin protein thus has activity similar to that of Co-Mb. Our group used cobalt substitution to create a proteinderived H2 evolution catalyst.70 This catalyst was prepared from the heme−peptide complex known as microperoxidase-11 (MP-11), which is prepared from proteolysis of horse cytochrome c. MP-11 consists of a heme attached to an 11mer peptide through a Cys−X−X−Cys−His motif in which the two Cys residues form covalent bonds to the porphyrin and His coordinates the iron (Figure 7A). Substitution of iron with cobalt was performed, and the free amino groups were acetylated to give acetylated cobalt microperoxidase-11 (CoMP11-Ac). Although the catalytic center is not buried in a protein scaffold, the covalent attachment of the porphyrin cofactor to the peptide that characterizes cytochrome c is in place in this construct. The three-dimensional structure of the Cys−X−X−Cys−His peptide in cytochrome c remains intact in microperoxidases, providing a defined structure on the proximal side of the heme and maintaining the axial His−Fe bond through a range of conditions.71 As an electrocatalyst, CoMP11-Ac operates with near-quantitative Faradaic efficiency and reaches a TON of 2.5 × 104 over 4 h of controlled potential electrolysis (CPE), although at a high overpotential (852 mV). Interestingly, this miniprotein catalyst has significantly enhanced activity as measured by TOF (determined as TON/time) relative to that reported for other watersoluble cobalt porphyrin electrocatalysts,72−74 although the basis for this difference is not yet known. Mechanistic studies and the preparation of derivatives are needed to better understand this system. A significant limitation of CoMP11Ac is its poor stability under electrocatalytic conditions because degradation is seen after 15 min. A high TON is measured despite the degradation because of the rapid rate of H2 evolution. This observation has stimulated the search for means by which to stabilize CoMP11-Ac to potentially yield systems for H2 production capable of very high TON values.

4. ELECTROCATALYTIC H2 PRODUCTION FROM A COBALT CYTOCHROME C CoMP11-Ac is an active electrocatalyst for H2 evolution from water at neutral pH as measured by TON and TOF values, but its rapid deactivation is a significant shortcoming.70 Another limitation of CoMP11-Ac is that it is difficult to modify to introduce features such as proton shuttles because its porphyrin is highly solvent-exposed on the distal side. Furthermore, outside of the Cys−X−X−Cys−His segment, the peptide is flexible and poorly structured.76,77 To address these issues, we prepared a biomolecular catalyst with an active site similar to F

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Inorganic Chemistry CD, oxidized protein samples (10 μM) were in 50 mM sodium phosphate, pH 7.0, in a 0.100-cm-path-length quartz cell. Three scans were collected for each sample from 190 to 260 nm using a 1 nm step size, a 1 nm bandwidth, and 1 s point−1. A 1H NMR spectrum of 0.5 mM Ht-CoM61A in D2O at an uncorrected pH of 5.0 was collected on a Bruker Avance 500 spectrometer. A total of 512 scans were taken with a recycle time of 2.4 s, a spectral width of 24 ppm, and 32K points. Electrochemical Methods. Electrochemical experiments were performed with a 620D potentiostat (CH Instruments). A Ag/AgCl (1 M KCl) reference electrode, a platinum wire counter electrode, and a mercury working electrode were utilized for CPE and CV experiments. CPE was performed in a three-compartment cell (CH Instruments) with three equivolume chambers of 1.5 cm diameter and 6 cm height. The chambers are separated by 10-mm-diameter P5 (1.0−1.6 μm pore size) glass frits. Mercury was chosen as a working electrode because of its large cathodic window, thus minimizing the background in water. Its connection to the potentiostat was enabled through an insulated platinum wire. A hanging mercury drop electrode (HMDE; Institute of Physical Chemistry of the Polish Academy of Sciences) was used as the working electrode for CV experiments. A fresh mercury drop (surface area 0.038 cm2) was introduced for each experiment, and thorough mixing of the solution prior to data collection was performed. Electrochemical cells were maintained under nitrogen for data collection. For CPE experiments, the cell was purged with 80% N2/20% CH4, with CH4 serving as an internal standard for gas chromatography (GC) experiments. A gastight seal of each compartment was achieved using septa. During experiments, the pH was monitored using a VWR SB70P pH meter and a Mettler Toledo InLab semimicro pH probe. For pH titrations, small volumes of 5 M KOH were added to adjust the pH, with no more than a total of 20 μL added for each titration. Measurement of H2. To determine the moles of H2 produced after CPE, 100 μL of the headspace gas from the electrolysis cell was injected into a Shimadzu GC-17A gas chromatograph with a thermal conductivity detector and a molecular sieve 5-Å column (30 m × 0.53 mm). The ratio of the H2 peak to the CH4 peak was determined through manual integration. This value was then analyzed using a standard curve, which was developed from known injected volumes of H2 at 1 atm. The moles of H2 generated and the total charge passed during CPE (Q) were used to determine the Faradaic efficiency (FE) as follows:

secondary structure as expected, with minima at 208 and 222 nm. The spectrum in this region is very similar to the spectra of wild-type Ht-c552 and of Ht-M61A, indicating that neither M61A mutation nor metal substitution significantly perturbs the protein secondary structure (Figure S3). The 1H NMR spectrum of Ht-CoIIIM61A has narrow, well-dispersed resonances, indicating that the protein is folded with a well-defined tertiary structure (Figure S4). These results demonstrate that the protein refolded properly after the metal substitution procedure. Ht-ZnM61A was prepared for control experiments. It has a UV−vis absorption spectrum typical of a zincsubstituted cytochrome (Figure S1)87,91 with absorption maxima at 420, 547, and 583 nm, and its CD spectrum indicates high α-helical content, although slightly lower than those of Ht c-552, Ht-M61A, and Ht-CoIIIM61A (Figure S3). The activity of Ht-CoM61A as an electrocatalyst for H2 evolution was tested by performing CPE. First, short (1 min) experiments were performed on a 1.0 μM sample in 2 M KPi, pH 7.0, at a range of applied potentials, with the amount of charge passed increasing at lower potentials as expected (Figures S5 and S6). On the basis of these results, a potential of −1.45 V vs Ag/AgCl (1 M KCl), corresponding to an 800 mV overpotential for proton reduction to H2 at pH 7.0, was selected for longer CPE experiments. CPE of 0.1 μM HtCoM61A (2 M KPi, pH 7.0) was run at −1.45 V for 6 h under nitrogen, a period during which charge buildup occurs at a nearly constant rate (Figure S7). The H2 produced during the experiment was quantified by GC. In at typical CPE experiment, at the end of 6 h, 60 ± 10 μmol of H2 was evolved, yielding a Faradaic efficiency of 92 ± 8% (see eq 4). The TON calculated based on moles of catalyst present in the bulk solution was 1.1 × 105 mol of H2 (mol of catalyst)−1 over 6 h. To test the longevity of the catalyst, CPE was run for 24 h. A TON of 2.7 × 105 and a Faradaic efficiency of 96 ± 6% were determined. The rate of H2 evolution decreases after the first 6 h, although the catalyst remains active at the 24 h mark (Figure S7). To determine whether Ht-CoM61A is the catalytically active species, CPE was performed for 1 h on Ht-CoM61A and related derivatives: Ht-FeM61A, zinc-substituted Ht-ZnM61A both alone and in the presence of 10-fold CoCl2, and HtPM61A. Under these conditions, only Ht-CoM61A yields a substantial increase in charge passed and H2 evolved over the background (Figure 8). Furthermore, the lack of activity for HtZnM61A with 10-fold excess CoCl2 demonstrates that the cobalt porphyrin moiety rather than cobalt associated with the polypeptide is the catalytically active species. To further characterize the properties of Ht-CoM61A as a proton reduction catalyst, CV experiments were performed in aqueous buffer using a HMDE. Cyclic voltammograms of HtCoM61A were found to exhibit a pH-dependent catalytic wave (Figure 9), with an onset of ∼−1.30 V (Figure S8) and a peak of ∼−1.45 V vs Ag/AgCl (1 M KCl). The current increases at lower pH, as expected for proton reduction. The dependence of the electrochemical response on the Ht-CoM61A concentration also was assessed. At low catalyst concentrations (up to ∼0.5 μM), the catalytic peak current increases linearly with the catalyst concentration. However, at higher concentrations, saturation behavior is observed (Figures S9 and S10). One explanation for this behavior is adsorption of the catalyst onto the electrode surface as the concentration is increased. To test this hypothesis, a rinse test was performed using the mercury pool electrode. After 30 min of CPE of 1.0 μM Ht-CoM61A,

FE = [96485 × 2 × mol of H 2 (GC) × 100%]/[Q (CPE)] (4)

TON values were determined by dividing the moles of H2 produced by the moles of catalyst in the reaction volume of the CPE cell (5 mL). For long experiments, over 1 h, the catalyst concentration was kept low (∼0.1 μM) in order to prevent excessive pressure buildup. 4.2. Results. Substitution of iron with cobalt in HtCoM61A was verified with UV−vis absorption spectroscopy. The absorption spectrum of Ht-CoM61A as purified displays a Soret band at 421 nm and Q bands at 533 and 567 nm (Figure S1), values characteristic of a cobalt(III) porphyrin species.89,90 Upon reduction, a spectrum consistent with a cobalt(II) porphyrin species with absorption maxima at 399 and 546 nm was obtained (Figure S2).90 Far-UV CD spectra were collected to assess the protein secondary structure. The spectrum of HtCoM61A is consistent with the presence of an α-helical G

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Inorganic Chemistry

is more than a 10-fold increase over that of CoMP11-Ac. This finding reveals that surrounding the cobalt porphyrin active site with polypeptide significantly enhances its robustness as an electrocatalyst. A second limitation of CoMP11-Ac is that it operates at a high overpotential. Previous studies were performed at an overpotential of 852 mV, which was determined to be the applied overpotential at which a maximum H2 evolution rate is achieved. For Ht-CoM61A, the corresponding value is 830 mV, which is nearly unchanged. Some cobalt catalysts such as the cobaloximes that have proton shuttles operate at significantly lower overpotentials.92,93 The engineering of this protein catalyst to introduce similar proton shuttles may yield a more efficient catalyst. The heme in Ht c-552 is buried in the polypeptide, and examination of its structure indicates that there is not an obvious route for water or protons to enter the active site other than structural fluctuations or partial unfolding.75 Future work will introduce mutations predicted to place protonatable groups near the cobalt center and between the heme and solvent. The interaction of the Ht-CoM61A with the mercury electrode is significantly different from that of CoMP11-Ac, which is not surprising when a full-length protein is compared with a small metalloporphyrin−peptide. There is no detectable adsorption of CoMP11-Ac to the electrode,70 despite the fact that porphyrins have been shown to adsorb to mercury electrodes.74 Apparently, the peptide attachment enhances the hydrophilicity sufficiently to maintain CoMP11-Ac in solution. In contrast, Ht-CoM61A shows significant adsorption. In fact, after the electrode is rinsed, the same rate of charge buildup as that observed before the rinse is maintained for minutes, suggesting that the catalysis occurs from an adsorbed species. A few minutes after the rinse, however, the rate of charge buildup drops. In contrast, the charge buildup is constant for many hours if there is Ht-CoM1A in solution (Figures S7 and S11). From these observations, we conclude that the Ht-CoM61A film on the electrode slowly exchanges with Ht-CoM61A in the solution. We also propose that, as the catalyst on the electrode is degraded or deactivated, it is replaced with fresh catalyst from solution to maintain a steady rate of H2 evolution over many hours. The finding that Ht-CoM61A adsorbs to mercury is not unexpected because proteins are known to adsorb to mercury through hydrophobic interactions with interaction times in the range of seconds.94,95 A concern with observing catalysis from an adsorbed species is that a degradation product such as cobalt particles rather than the intact cobalt porphyrin may be the catalytically active species. However, mercury is known to inhibit the activity of colloidal catalysts.96,97 Furthermore, mercury forms an amalgam with cobalt.98 Thus, measurement of the activity on a mercury electrode rules out these complications. Furthermore, the lack of H2 evolution activity for Ht-ZnM61A with 10-fold excess of CoCl2 (Figure 8) also supports the activity of a cobalt porphyrin specifically rather than a degradation product or some undefined cobalt− polypeptide complex. Cytochrome c transfers electrons to and from redox partners through a partially solvent-exposed heme edge. The amino acids near the exposed heme edge form a patch that is recognized by redox partners.99 The exposed heme edge of Ht c-552 is surrounded by hydrophobic amino acids, giving a hydrophobic patch for binding to redox partners, which is characteristic of cytochrome c in its structural class.75 Ht-

Figure 8. CPE of 1 μM Ht-CoM61A (purple), 1 μM Ht-FeM61A (blue), 1 μM Ht-ZnM61A (green), 1 μM Ht-ZnM61A and 10 μM CoCl2 (orange), 1 μM Ht-PM61A (black), and buffer only (red). In all experiments, the buffer used was 2 M KPi, pH 7.0, the working electrode was a mercury pool electrode, and the applied potential was −1.45 V vs Ag/AgCl (1 M KCl).

Figure 9. Cyclic voltammograms (100 mV/s) of 1.0 μM Ht-CoM61A in 25 mM KPi and 1 M KNO3 as a function of the pH.

the working electrode compartment was rinsed several times with buffer without disturbing the mercury pool. A fresh batch of buffer was then added to each compartment of the cell, and CPE was performed again. Although the UV−vis spectrum of the solution above the electrode showed no detectable catalyst within the fresh buffer solution, a substantial amount of H2 was evolved during the course of the experiment. In fact, the initial rate of charge buildup from the rinsed electrode is similar to that seen for the original solution, although the rate declines after 5 min (Figure S11). Finally, the scan rate dependence of the Ht-CoM61A cyclic voltammograms was determined (100− 1500 mV/s). For CV performed on 1.0 μM Ht-CoM61A in 2 M KPi, pH 7, no scan rate dependence was observed (Figure S12). 4.3. Discussion. We have demonstrated that substituting iron with cobalt in a thermophilic cytochrome c mutant yields a H2-evolving electrocatalyst capable of a TON > 270000. A goal of this work was to engineer a biomolecular catalyst with an active site similar to that of CoMP11-Ac but with greater longevity. The electrocatalytic activity of Ht-CoM61A shows minimal decline over ∼6 h, and it remains active for ∼24 h. In contrast, CoMP11-Ac shows a significant decline in activity after 20 min and is active only for a few hours. Furthermore, the TON for H2 evolution by Ht-CoM61A exceeds 270000, which H

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Inorganic Chemistry CoM61A thus may interact with the mercury electrode at this site. However, it is possible that the protein unfolds partially or extensively on the mercury surface. Nevertheless, it remains active and displays greatly enhanced longevity relative to CoMP11-Ac. CV of Ht-CoM61A in a 2 M KPi buffer shows no scan rate dependence in the range of 100−1500 mV s−1. This observation is consistent with electrochemistry on an adsorbed catalyst operating with an excess of substrate. Interestingly, lowering buffer concentration to 10 mM KPi reveals a scan rate dependence (Figures S13 and S14). The dependence is complex, but we propose that diffusion of buffer as the proton donor to the catalyst plays a role in determining the scan rate dependence at low buffer concentration. More studies are needed to better understand this behavior. Mercury is not generally a biocompatible electrode because of its hydrophobicity, although it was utilized here for its large cathodic window and to facilitate comparison to published work.70 Future studies will make use of more biocompatible electrodes such as pyrolytic graphic edge electrodes.100−102 However, previous work has shown that, even on pyrolytic graphite, cytochrome c can occupy more than one conformation, and thus perturbation of the protein structure by surface binding remains a concern.103,104 Another approach underway is the activation of Ht-CoM61A by pairing it with photosensitizers for photoinduced electron transfer. In addition to preserving the structure and fold of the protein and its variants, an advantage of this approach is that it accomplishes the storage of light energy in the form of H2. Furthermore, employing Ht-CoM61A in a photocatalytic system will facilitate comparison of its activity to other protein-based catalysts for H2 evolution because most of these systems operate in light-driven systems.

standing of how the structure and dynamics of both the active site and the polypeptide influence the reaction mechanism is needed. Using protein engineering and protein chemistry to manipulate electron- and proton-transfer pathways may provide unique insight into how the protein structure affects the assembly of electrons and protons to make H2. For example, by judicious placement of various photoactive electron donors on defined positions on the protein catalyst, the rates of electron injection can be controlled and studied.105 Altering the nature of the electron donor can influence the driving force. Furthermore, mutations at and near the metal site will influence its contribution to the reorganization energy for electron transfer. In general, the field would benefit from making more use of the wealth of fundamental knowledge that has been gained on long-range electron transfer in biological systems.53,105,106 Finally, a powerful approach to preparing biomolecular catalysts that has not yet been applied to prepare an active H2 evolution catalyst is computation-based metalloprotein design.107,108 Collaborations between experts in protein folding and design and experts in redox catalysis as well as catalyst synthesis could open new and exciting pathways forward.

5. CONCLUSIONS AND PERSPECTIVE Ht-CoM61A is a refinement of the CoMP11-Ac catalyst, which was identified to have two primary weaknesses. One is its rapid deactivation, and the other is its high overpotential. We hypothesized that Ht-CoM61A, with its active site protected by polypeptide, may remain active for a longer period of time, and we did find this to be the case. This finding is in line with the results from other laboratories on photocatalytic systems. For example, CoP bound to Mb68 or cytochrome b562 polypeptide73 remains active longer than the same species in solution. In another example, a [FeFe] mimic bound to nitrobindin shows greater longevity and higher TON than this same mimic bound to smaller and/or less-structured polypeptides.61 These results do suggest that burying a catalytic site within a protein matrix can enhance longevity, although the basis for this effect is not known. While the longevity of Ht-CoM61A is very good, its overpotential is high and nearly unchanged from that of CoMP11-Ac. It is interesting that the overpotential is similar for the cobalt porphyrin site both when highly solvent-exposed and when surrounded by polypeptide, and this result may indicate that it will be difficult to lower it through catalyst modification. However, efforts to do so by altering the active site structure and changing the conditions are underway. The development of protein-based systems for H2 evolution is still at an early stage. Challenges that need to be met are preparing structurally defined and structurally characterized catalysts, obtaining detailed mechanistic information, and systematically changing properties such as the overpotential through protein engineering. In particular, a greater under-

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.5b02054. Absorption, CD, and NMR spectra, additional electrochemical data, and a table reporting the properties of photocatalytic systems discussed herein (PDF)

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AUTHOR INFORMATION

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (Grant DEFG02-09ER16121).

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REFERENCES

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DOI: 10.1021/acs.inorgchem.5b02054 Inorg. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.inorgchem.5b02054 Inorg. Chem. XXXX, XXX, XXX−XXX