Letter pubs.acs.org/JPCL
Nickel-Substituted Rubredoxin as a Minimal Enzyme Model for Hydrogenase Jeffrey W. Slater and Hannah S. Shafaat* Department of Chemistry and Biochemistry and Ohio State Biochemistry Program, The Ohio State University, 100 W 18th Avenue, Columbus, Ohio 43210, United States S Supporting Information *
ABSTRACT: A simple, functional mimic of [NiFe] hydrogenases based on a nickelsubstituted rubredoxin (NiRd) protein is reported. NiRd is capable of light-initiated and solution-phase hydrogen production and demonstrates high electrocatalytic activity using protein film voltammetry. The catalytic voltammograms are modeled using analytical expressions developed for hydrogenase enzymes, revealing maximum turnover frequencies of approximately 20−100 s−1 at 4 °C with an overpotential of 540 mV. These rates are directly comparable to those observed for [NiFe] hydrogenases under similar conditions. Like the native enzymes, the proton reduction activity of NiRd is strongly inhibited by carbon monoxide. This engineered rubredoxin-based enzyme is chemically and thermally robust, easily accessible, and highly tunable. These results have implications for understanding the enzymatic mechanisms of native hydrogenases, and, using NiRd as a scaffold, it will be possible to optimize this catalyst for application in sustainable fuel generation.
H
ydrogen is considered an ideal fuel of the future for many reasons, though anthropogenic hydrogen production remains impractical and unsustainable.1 By contrast, Nature employs specialized metalloenzymes called hydrogenases to process hydrogen; these enzymes have been of great interest for fundamental research and design of bioinspired synthetic molecular catalysts over many decades.2−12 It has become increasingly apparent that noncovalent elements play critical roles in catalysis,13,14 with the native hydrogenase enzyme providing a highly conserved network of secondary and tertiary sphere interactions.7,15,16 Considering the challenge of preserving specific interactions in a synthetic compound or a small peptide, we have elected to pursue the development of a new hydrogenase enzyme using rubredoxin (Rd), a natural metalloprotein with a well-defined fold and coordination environment, as a scaffold. Rubredoxins are small (5−6 kDa), robust electron transfer (ET) proteins that bind iron in a tetrathiolate, mononuclear site (FeRd). These proteins are thermally and chemically stable,17,18 well-structured in solution despite their small size, and are easily expressed and purified in high yields using standard techniques (see Supporting Information and Figures S1−S4 for details).19 The active-site iron can be exchanged for a number of other metal centers, including nickel, and the protein remains air-stable.20 Because the primary coordination sphere of nickel-substituted rubredoxin (NiRd) mimics the primary coordination sphere of the only redox-active metal in the [NiFe] hydrogenases (Figure 1),2,3 these proteins serve as a minimal structural model for the native system. NiRd is also an ideal platform from which to identify critical molecular components for effective catalysis and engineer new hydrogenase enzymes. Additional support for this approach was © 2015 American Chemical Society
Figure 1. To-scale ribbon diagrams (top) and active-site structures (bottom) of (A) Desulfovibrio vulgaris Miyazaki F [NiFe] hydrogenase (PDB ID 1H2R) and (B) Desulfovibrio desulfuricans Rd (PDB ID 6RXN). Active site of NiRd is modeled from FeRd structure.
Received: August 10, 2015 Accepted: September 2, 2015 Published: September 2, 2015 3731
DOI: 10.1021/acs.jpclett.5b01750 J. Phys. Chem. Lett. 2015, 6, 3731−3736
Letter
The Journal of Physical Chemistry Letters
To confirm the production of hydrogen, solution-based assays were conducted. A mixture of NiRd, ascorbate, and [Ru(bpy)3]2+ as a photosensitizer was irradiated with 4.5 mW of 447.5 nm light at 4 °C, and gas chromatography (GC) headspace analysis was carried out as a function of time (Figure 3). Photoinitiated hydrogen production is observed with an initial turnover frequency (TOF) of ∼0.5 min−1 (80.6 ± 14.4 nmol/min/mg catalyst). At 25 μM catalyst loading, hydrogen evolution reaches a maximum within an hour, with a total turnover number (TON) of 32 over this time (Table ST1). At lower catalyst concentrations, similar levels of activity (97.3 ± 27.9 nmol/min/mg catalyst) persist over 8 h, with a TON > 100 (Table ST2). The observed concentration dependence suggests bimolecular quenching is responsible for the decrease in NiRd activity over time; however, over 90% of the sample can be recovered following the assay (Table ST3 and Figure S11), suggesting that the inactivation process does not degrade the protein active site. Traditionally, “dark” solution-based assays for quantifying hydrogen evolution by hydrogenases use dithionite as a sacrificial electron donor (E°′ = −0.66 V vs NHE at pH 7)27 and methyl viologen as a redox mediator.20,28,29 However, the low onset potential for electrocatalysis by NiRd necessitates a stronger reducing agent than dithionite; indeed, no significant hydrogen evolution was observed by GC analysis using only dithionite or dithionite with methyl viologen. Titanium(III) citrate is an aqueous reducing agent with a lower reduction potential than dithionite (E°′ = −0.85 vs NHE at pH 7).30 Considering this, solutions of 2.5 μM and 25 μM NiRd were stirred at room temperature in the presence of excess titanium(III) citrate (Figure S12). The headspace volume sampled by GC analysis indicated that 25.5 ± 3.2 nmol H2/ min/mg NiRd were generated over 8 h for the lower concentration samples, corresponding to a TOF of ∼0.1 min−1 with a TON > 300 (Table ST4). There was no indication of catalyst degradation over that time, and nearquantitative recovery of NiRd was achieved (Table ST3). However, the solution potential and baseline levels of hydrogen were found to be highly sensitive to minor perturbations in preparation protocols and varied across commercial suppliers and between batches. Consistent with these observations, an electrochemical and spectroscopic study on aqueous solutions
found in an early report that suggested potential for weak hydrogenase activity in metal-substituted rubredoxins that were isolated from hydrogenase-containing, sulfate-reducing bacteria.20 Here, we report that recombinantly expressed NiRd from Desulfovibrio desulfuricans ATCC 27774 shows high activity for hydrogen production in solution, upon photoinduction, and when adsorbed to an electrode. Cyclic voltammograms (CVs) obtained by protein film electrochemistry (PFE) of NiRd and FeRd are shown in Figure 2. Signals due to a fully reversible FeIII/II couple are observed at
Figure 2. CVs comparing surface-adsorbed NiRd, FeRd, and the bare pyrolytic graphite electrode in acetate buffer, pH 4.5, 4 °C, scan rate = 50 mV/s. (Inset) Expanded view of region showing the FeIII/II reversible couple.
−34 mV vs NHE for FeRd, which exhibits well-behaved electrochemical signals consistent with a properly folded, adsorbed protein with an electroactive coverage (Γ) of 25 ± 10 pmol/cm2 (Figures S5−S9).21−26 FeRd ET kinetics were determined by monitoring the positions of the oxidative and reductive waves as a function of scan rate and fitting the data with Butler−Volmer theory, giving a rate constant of 80 s−1 (Figures S7).23 In contrast to the reversible one-electron signal seen for FeRd, the voltammogram of NiRd displays large catalytic currents beginning at approximately −800 mV vs NHE. This cathodic current is buffer independent (Figure S10) and has been tentatively attributed to catalytic proton reduction activity.
Figure 3. Photoinduced hydrogen evolution by NiRd with (A) 25 μM and (B) 2 μM catalyst along with 1 mM [Ru(bpy)3]2+, 100 mM ascorbate, and 1 M potassium phosphate, pH 6.5 at 4 °C. Traces correspond to NiRd (green), FeRd (red), no catalyst (black), and NiRd lacking [Ru(bpy)3]2+ (gray). 3732
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The Journal of Physical Chemistry Letters
observed for the [NiFe] hydrogenases.37 The absence of a noncatalytic ET signal across all pHs and scan rates (Figure S15) suggests that initial reduction is the rate-limiting step, with subsequent protonation and electron transfer steps occurring within the catalytic wave.37 Thus, the onset of catalysis can be estimated to occur at the potential of initial reduction, with an overpotential (η) of 540 mV between pH 3 and pH 5. While this process is less energetically efficient than the native enzymes, the NiRd overpotential is comparable to that required by many other synthetic hydrogen production catalysts based on earth-abundant transition metals operating in aqueous solutions, and lower than those recently determined for artificial catalysts within protein scaffolds.5,13,34,39,40 To accurately simulate the NiRd CVs, it must be taken into account that the signals are nonideal, lacking the characteristic sigmoidal shape seen for well-behaved catalysts. The electrocatalytic model has been adapted to account for a distribution of protein orientations on the surface, which causes a dispersion of interfacial ET rate constants (kET) and gives rise to a residual slope in the catalytic current at high overpotentials.41 In the limiting case, this distribution suggests that a regime in which kET is much greater than kcat may not be accessible; slow interfacial ET can also give rise to this phenomenon. This consideration explains the shape of the CVs of highly efficient enzymes such as hydrogenase and carbon monoxide dehydrogenase; its requisite inclusion to model the voltammograms of NiRd highlights the similarities between NiRd and the [NiFe] hydrogenase.37,41−43 As with the native enzymes, assessing parameters such as the TON, TOF, and η based on traditional metrics is not possible. Nevertheless, limiting values of the electrocatalytic TOF can be estimated. If it is assumed that the electroactive coverages of FeRd and NiRd are similar, evaluating the catalytic current at −100 mV beyond the initial reduction potential (iE(O/I)‑100mV) gives a TOF of 20 s−1 as a lower limit (Figure S16). When the limiting currents found from the electrocatalytic simulations (ilim) are used, the TOF at pH 4.5 is calculated to be 100 s−1. That these values are within an order of magnitude of each other demonstrates consistency between the qualitative analysis of the raw voltammograms and semiquantitative analysis via electrocatalytic simulations. Additional support for the higher calculated NiRd TOF of 100 s−1, relative to the estimated lower limit of 20 s−1, is derived from fitted values for kcat/kET,max, which at low pH are found to be approximately 1 (Table ST5). The observed FeRd ET rate of 80 s−1 is much slower than solution-phase selfexchange rates,44 consistent with the observation that PFE kinetics can be dominated by the interfacial component rather than intramolecular rearrangement.23 Thus, it is reasonable to estimate similar values of kET,max for NiRd and FeRd. Given this assumption, the TOF can be estimated at ∼80 s−1, comparable to the TOF of 100 s−1 presented above. The values of ilim and kcat/kET,max report on different portions of the voltammogram, reflecting high and low driving force, respectively. The agreement between parameters obtained from these distinct regions of the simulated voltammogram underscores the robustness of the electrocatalytic model. Importantly, the catalytic rates of proton reduction by NiRd are directly comparable to the TOF for electrocatalytic hydrogen production by the native [NiFe] hydrogenase, which at 5 °C has been reported to be 50 s−1.37 As an additional point of comparison between NiRd and the [NiFe] hydrogenases, the response of the electrocatalytic signal to carbon monoxide exposure was investigated. It is well-
of titanium(III) citrate has established that the redox chemistry is only quasi-reversible, and the speciation is not well-defined.31 Thus, at this point in time, we restrict our analysis to a qualitative discussion, concluding that NiRd is also active for solution-phase hydrogen evolution with a low-potential electron donor. In both assays, the NiRd activity is lower than many of the [NiFe] hydrogenases, although the activities of the native enzymes are also highly strain-dependent.29 Corresponding rates for photoinduced activity of native [NiFe] hydrogenases using ruthenium photosensitizers range from 2 to 540 min−1.32,33 However, NiRd photocatalytic rates are directly comparable to those seen in previously reported semisynthetic enzyme systems, with TONs that are within an order of magnitude of those optimized systems.34,35 Additionally, it is known that the native hydrogenase activity is significantly hindered in solution relative to that observed when immobilized on an electrode.36 To further probe the electrocatalytic activity of surfaceadsorbed NiRd, CV experiments were carried out from pH 3.0 to pH 7.0 (Figures 4 and S13). Under acidic conditions, the
Figure 4. Catalytic voltammograms of surface-adsorbed NiRd at varying pH, 4 °C, scan rate = 25 mV/s, rotation rate = 1500 rpm. The average of the anodic and cathodic waves is shown, with experimental data displayed by markers (only 1 experimental point in every 100 collected is plotted) and simulated voltammograms shown as solid lines. (Inset) Expanded catalytic region for pH 5.5−7.0 (only 1 experimental point in every 10 collected is shown).
catalytic current shifts toward more negative potentials as the pH is increased. Above pH 5, catalysis becomes independent of pH (Figure 4, inset). Using expressions derived for a twoelectron, two-proton electrocatalytic process,37 the CVs of NiRd across multiple pHs can be accurately simulated (Figure 4). The complete set of parameters obtained from these simulations along with a detailed discussion of the simulation procedure is presented in the Supporting Information (Table ST5). By assessing the individual reduction potentials (EO/I and EI/R), limiting current (ilim), and ratios between apparent catalytic and ET rate constants (kcat/kET), insight into the electrocatalytic mechanism can be obtained. Even in the case of NiRd, which exhibits relatively featureless CVs, accurate simulations can constrain the possible mechanisms. Specifically, a linear relationship is observed between the initial reduction potential, EO/I, and pH from pH 3 to pH 5, with an observed slope of −67 mV/pH unit (Figure S14). This is consistent with a proton-coupled electron transfer process38 and similar to that 3733
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reduction. Catalytic hydrogen evolution is observed in aqueous solutions using photochemical and chemical reducing agents and with a protein film adsorbed on the surface of an electrode. Initial estimates suggest catalytic rates for NiRd of approximately 20−100 s−1; the overpotential of 540 mV is similar to those observed with many types of model compounds operating in aqueous solutions.4,5 While there are molecular platforms, including the nickel phosphine catalysts with pendant amines pioneered by Dubois,12,47,48 that can operate at high catalytic rates with essentially no overpotential,13,40 the original compounds were less effective.49,50 Only with iterative optimization of these platforms were catalysts developed that exhibited both low overpotentials and high rates.13,14,51 An analogous process can now be pursued with NiRd. For example, functional side chains, such as those conserved around the active site of the [NiFe] hydrogenases,7,15 can be installed into the secondary coordination sphere to provide proton shuttles and tune the reduction potential. Through rational design and optimization, it should be possible to create highly active artificial hydrogenase enzymes. Importantly, the electrocatalytic behavior of NiRd closely resembles that of the [NiFe] hydrogenase, and the rates of hydrogen production are directly comparable to those of the native enzyme. Further studies are currently underway to elucidate the catalytic mechanism, obtain structural information on intermediates, and enhance enzymatic function and robustness. NiRd is a simple and easily accessible functional and structural model for the [NiFe] hydrogenases and represents the first generation of a new class of hydrogenase mimic. Studies on NiRd provide an opportunity to address one of the remaining questions about the [NiFe] hydrogenase; specifically, identifying the role that the Fe center plays in the activity of the enzyme. Development of this platform will also provide fundamental insight into critical requirements for effective molecular catalysts, with future potential for application in solar fuel generation.
established that most [NiFe] hydrogenases are strongly inhibited by CO, and prior solution-phase work on NiRds also suggested high levels of sensitivity to CO for both protium/deuterium exchange and H2 evolution.2,3,20,45 In this work, while no interaction was observed upon exposure of resting-state NiIIRd to CO in solution, introduction of CO into the electrochemical cell headspace causes the catalytic current to decrease rapidly (Figure 5). The inhibition curve likely
Figure 5. Cyclic voltammograms of surface-adsorbed NiRd in acetate buffer at pH 4.5, 4 °C, upon introduction of CO or N2 gas. Scan rate = 100 mV/s, rotation rate = 1500 rpm. (Inset) Maximum current monitored over time for CO and N2 introduced into the cell headspace. Dashed line shows corrected signal accounting for film loss.
represents binding of CO to the reduced Ni center, preventing catalysis.46 Unlike the [NiFe] hydrogenases,2 flushing the electrochemical cell with nitrogen following CO exposure does not restore NiRd catalytic activity. We speculate that the reduction potential of the CO-bound species is shifted beyond the range of the experiment, rendering binding essentially permanent under the conditions employed. It was further observed that the NiRd catalytic signal is independent of electrode rotation rate (Figure S17). This behavior differs from that of the [NiFe] hydrogenases; however, in the native enzyme, the strong dependence on rotation rate is generally thought to reflect limited product release rather than substrate diffusion.37 The moderate overpotential observed for NiRd precludes hydrogen oxidation,43 rendering this an ideal system for H+ reduction even under conditions of high hydrogen concentrations. Additional control experiments were performed to probe whether electroplating of nickel from the protein onto the electrode surface could be responsible for the catalytic current observed in the cyclic voltammetry experiments. Following a set of CV experiments on immobilized NiRd, the pyrolytic graphite electrode was rinsed thoroughly with 50 mM Tris buffer, pH 8.0. The rinsed electrode showed no residual electrocatalytic signal above the baseline capacitative current, suggesting complete dissociation of NiRd from the electrode surface (Figure S18). An analogous control experiment was performed on FeRd, and similar protein desorption was observed (Figure S18). In summary, we report that a nickel-substituted rubredoxin protein is an effective artificial hydrogenase for proton
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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.jpclett.5b01750. Description of materials and methods along with supplemental figures and additional control experiments (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by The Ohio State University Department of Chemistry and Biochemistry, an OSU Institute for Materials Research Facility Grant, and the National Science Foundation under grant CHE-1454289. The authors would like to thank Sabrina Cirino, Haleigh Monaco, and Joshua Olive for assistance with sample preparation, Claudia Turro for the use of her gas chromatograph and LEDs, Dmitri Kudryashov for the use of his circulator, and Christopher Jaroniec, David Heisler, and members of the Shafaat and Cowan laboratories for helpful conversations. 3734
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DOI: 10.1021/acs.jpclett.5b01750 J. Phys. Chem. Lett. 2015, 6, 3731−3736
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DOI: 10.1021/acs.jpclett.5b01750 J. Phys. Chem. Lett. 2015, 6, 3731−3736
