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Light-driven dinitrogen reduction catalyzed by a CdS:nitrogenase MoFe protein biohybrid

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Science  22 Apr 2016:
Vol. 352, Issue 6284, pp. 448-450
DOI: 10.1126/science.aaf2091

Enzymes make fertilizer with sunlight

Nitrogenase enzymes catalyze the biological production of fixed nitrogen. Because this is not enough to sustain modern agriculture, industrial fertilizers containing ammonia are produced via the energy-intensive Haber-Bosch process. Brown et al. developed a way to use nitrogenase enzymes from nitrogen-fixing bacteria to make ammonia in vitro without other biological steps or high-energy inputs. Light-activated CdS nanorods provided electrons to the FeMo nitrogenase enzyme to reduce nitrogen and produce ammonia at rates up to 64% of biological nitrogen fixation. These nanoparticle-protein complexes show the potential for solar-driven ammonia production.

Science, this issue p. 448

Abstract

The splitting of dinitrogen (N2) and reduction to ammonia (NH3) is a kinetically complex and energetically challenging multistep reaction. In the Haber-Bosch process, N2 reduction is accomplished at high temperature and pressure, whereas N2 fixation by the enzyme nitrogenase occurs under ambient conditions using chemical energy from adenosine 5′-triphosphate (ATP) hydrolysis. We show that cadmium sulfide (CdS) nanocrystals can be used to photosensitize the nitrogenase molybdenum-iron (MoFe) protein, where light harvesting replaces ATP hydrolysis to drive the enzymatic reduction of N2 into NH3. The turnover rate was 75 per minute, 63% of the ATP-coupled reaction rate for the nitrogenase complex under optimal conditions. Inhibitors of nitrogenase (i.e., acetylene, carbon monoxide, and dihydrogen) suppressed N2 reduction. The CdS:MoFe protein biohybrids provide a photochemical model for achieving light-driven N2 reduction to NH3.

The reduction of dinitrogen (N2) to ammonia (NH3) is the single largest input of fixed nitrogen (N) into the global biogeochemical cycle. Although the overall reaction releases energy, the cleavage of the nitrogen-nitrogen triple bond has a very large activation barrier. In the industrial Haber-Bosch process, NH3 is produced via a dissociative reaction involving coactivation of dihydrogen (H2) and N2 over an Fe-based catalyst (1). The H2 used for the reaction is produced by steam reforming of natural gas and results in coproduction of appreciable amounts of CO2. The energy required (>600 kJ mol–1 NH3) to achieve the high temperatures (500°C) and pressures (200 atm) necessary to drive the reaction is also largely derived from fossil fuels (2, 3).

In nitrogen-fixing bacteria, the enzymatic reduction of N2 to NH3 is catalyzed by nitrogenase enzymes and proceeds via the hydrogenation of N2 through metal-hydride intermediates rather than from reaction with H2 (4). The Mo-dependent nitrogenase is a multiprotein complex composed of MoFe and Fe proteins, named after the metals in their active sites. Although nitrogenase functions at room temperature (25°C) and pressure (1 atm), it requires a large input of chemical energy provided by the hydrolysis of adenosine 5′-triphosphate (ATP) (Fig. 1A) (4). A minimum of 16 moles of ATP (ΔG° = –488 kJ mol−1 or 5 eV mol–1 of N2 reduced) is required to reduce N2 to NH3. During catalysis, the Fe protein associates with and dissociates from the MoFe protein, resulting in the eight sequential electron transfer/ATP hydrolysis events required to generate 1 mole of NH3 (5). Reducing equivalents accumulate at the catalytic site FeMo cofactor (FeMo-co) as Fe-hydrides (6), which directly participate in conversion of N2 to 2 moles of NH3 with an obligatory stoichiometric reduction of two protons to make H2 (Fig. 1A) (4, 7).

Fig. 1 Reaction scheme for N2 reduction to NH3 by nitrogenase and the CdS:MoFe protein biohybrids.

(A) reaction catalyzed by nitrogenase Fe protein (homodimer represented in green; MgATP binding site, orange sphere; [4Fe–4S] cluster, brown square) and MoFe protein (α2β2 tetramer represented in gray and purple; FeMo-co, red hexagon; [8Fe–7S] P cluster, blue sphere). Hydrolysis of 16 ATP by Fe protein (Em = –0.42 V) is required for the sequential transfer (signified by the equilibrium arrow) of eight electrons (e) to MoFe protein (Em = –0.31 V) for catalytic reduction of N2 to 2NH3 and 1H2 (18). (B) The reaction catalyzed by CdS:MoFe protein biohybrids (measured product ratios were 1NH3/10H2, with n ≈ 98 absorbed photons; table S8). Under illumination, photon absorption (405 nm photon = 3.06 eV) by CdS nanorods (orange; lowest-energy transition, Eg = 2.72 eV; fig. S1) generates electrons (E = –0.8 eV) and holes (E = +1.9 eV) (1517), where direct electron injection from CdS into MoFe protein (blue arrow) is thermodynamically favored (ΔE = 0.5 V). The ground state of the CdS nanorod is regenerated by the oxidation of a sacrificial electron donor (D), such as HEPES (Em = + 0.8 V versus SHE) (22).

Low-potential chemical donors or photoexcited chromophores can directly deliver electrons to the MoFe protein. Complexes between MoFe protein and the low-potential donor Eu(II)-L (8, 9) or Ru-photosensitizers (10, 11) support the catalytic reduction of protons or nonphysiological C or N substrates (e.g., C2H2, HCN, N2H4, N3). However, these complexes are unable to catalyze N2 reduction, and rates for nonphysiological substrates are low (up to 8.5 min–1) compared to physiological reaction rates (e.g., 500 min–1 for C2H2 reduction). In the case of Ru-photosensitizers, it was shown that the Ru conjugate was unstable, resulting in the loss of photocatalytic rates and low quantum yields (QY ≤ 1%) (10, 11).

Here, we examined N2 reduction by the MoFe protein when it is adsorbed onto CdS nanocrystals to form biohybrid complexes (12). Semiconductor nanocrystals are quantum-confined materials with size-tunable photoexcited electron and hole energy levels (13). Different nanocrystalline materials were tested (table S1), and CdS nanorods (d ≈ 38 ± 5 Å, l ≈ 168 ± 16 Å; fig. S1) were observed to deliver photogenerated electrons to the MoFe protein with the highest enzymatic turnover. The size, shape, and surface electrostatics of the CdS nanorods complement the MoFe protein dimensions (d ≈ 69 Å, l ≈ 110 Å) and surface electrostatics to support self-assembly into complexes (14, 15). The lowest-energy transition of the CdS nanorods is in the visible region of the solar spectrum (Eg = 2.72 eV, λabsorption = 456 nm, fig. S1), and the reduction potential of the first photoexcited state transition, –0.8 V versus NHE (1517), is sufficiently negative to reduce the MoFe protein (–0.31 V) (18) to drive electron transfer for catalytic reduction of N2 to NH3 (Fig. 1B).

Photoexcitation of the CdS:MoFe protein biohybrids under a 100% N2 atmosphere resulted in the direct light-driven reduction of N2 to NH3 (Fig. 2, fig. S2 and tables S2 to S4). Transfer of low-potential electrons to the MoFe protein from the photoexcited CdS nanorod replaced ATP-coupled electron transfer by the Fe protein. The reaction required a sacrificial electron donor, HEPES, which produced a high turnover frequency (TOF) with a low background compared to other donors (table S2). Control reactions that lacked a key component (e.g., HEPES, CdS, light, or a functional MoFe protein) or utilized apo-MoFe protein that lacks FeMo-co did not produce NH3 (tables S3 and S5). Illumination under ~3.5 mW cm–2 of 405-nm light led to peak NH3 production rates of 315 ± 55 nmol NH3 (mg MoFe protein)–1 min–1 at a TOF of 75 min–1 (Fig. 2 and table S6). The values correspond to 63% of the NH3 production [500 nmol NH3 (mg MoFe protein)–1 min–1], and TOF (119 min–1) catalyzed by the Fe protein and ATP-dependent reaction under optimal conditions (table S6). The estimated QY of 3.3% for conversion of absorbed photons to NH3 (QY = 23.5% for the coproduction of NH3 and H2; tables S7 and S8) is higher than reported for other nonphysiological reactions (10, 11). N2 reduction persisted for up to 5 hours under constant illumination (Fig. 2, inset; tables S9 and S10) with a turnover number (TON) of 1.1 × 104 mol NH3 (mol MoFe protein)–1. This indicates that the MoFe protein in CdS:MoFe protein biohybrids can function at rates comparable to physiological TOF by nitrogenase.

Fig. 2 Photochemical N2 reduction to NH3 by CdS:MoFe protein biohybrids.

The TOF of catalytic reduction of N2 to NH3 was measured under 100% N2 (red). The effects of MoFe protein inhibitors on the TOF are shown for 10% of either H2 (cyan), carbon monoxide (CO, green), or acetylene (C2H2, brown) in a bulk phase of 90% N2. TOF for the CdS:MoFe protein biohybrids under 100% Ar (gray) is shown as a negative control for comparison. Measured values were taken after 2 hours of illumination at 25°C for reactions comprising 1:1 molar ratios of CdS nanorods and MoFe protein tetramer. Data are means of N = 4 independent measurements ± SD calculated by standard error propagation. (Inset) Time course of NH3 production by CdS:MoFe protein biohybrids under 100% N2 (12). TON = 1.1 × 104 mol NH3 (mol MoFe protein)–1 (table S10).

The mechanism of N2 reduction by the MoFe protein coproduces H2 (Fig. 1), which was also observed as a coproduct during CdS:MoFe protein photocatalytic N2 reduction (fig. S3 and tables S4 and S5). This supports a mechanism of N2 reduction by the CdS:MoFe protein biohybrids that is analogous to the mechanism of MoFe protein:Fe protein catalysis. CdS inhibition of Fe protein–dependent catalysis (table S11) indicates that CdS binds at or near the Fe protein binding site on MoFe protein (Fig. 1B); however, it is not known whether the P cluster serves as an intermediate in electron transfer during photocatalysis.

Additional evidence that the N2 reduction reaction occurs at FeMo-co of the MoFe protein was observed with known inhibitors of Mo-dependent nitrogenase activity. Acetylene (C2H2), carbon monoxide (CO), and H2 are all known to specifically inhibit the N2 reduction reaction at FeMo-co. Acetylene acts as a substrate to inhibit N2 and proton reduction at FeMo-co (19, 20). In contrast, CO inhibits N2 reduction by blocking the N2 binding site at FeMo-co, but proton reduction to H2 is unaffected (21). The addition of either H2, CO, or C2H2 at 10% to a 90% N2 gas phase decreased the N2 reduction rates by CdS:MoFe protein biohybrids to the background levels observed with apo-MoFe protein (Fig. 2 and tables S12 and S13). The results are consistent with the effect of these inhibitors on preventing MoFe protein catalysis in the Fe protein, ATP-driven physiological reaction. Photochemical H2 production by CdS:MoFe protein biohybrids was also inhibited by 10% C2H2, but only slightly decreased under 10% CO compared to rates under 100% N2 (fig. S3). Consistent with N2 being a substrate of CdS:MoFe protein biohybrids, the rates of H2 production were 25% higher when N2 was replaced with 100% argon (fig. S3). Together, the inhibition results are consistent with photocatalysis by CdS:MoFe protein biohybrids occurring at the FeMo-co site of the MoFe protein by a mechanism that is similar to that for the Fe protein, ATP-coupled reaction.

Although the CdS nanorods have a low photoexcited state potential (–0.8 V versus NHE), other reductants, such as Eu(II)-L, have lower potentials (as low as –1.2 V versus NHE), yet only the CdS nanorods support N2 reduction by MoFe protein. This indicates that some aspect of the nanorods, other than photochemical driving force alone, contributes to the achievement of N2 reduction. One possible explanation is the rapid delivery of successive electrons due to strong light absorption by the CdS nanorods, which could allow achievement of the four-electron reduced FeMo-co state (E4) that is required for N2 binding and reduction (4, 6). Slow accumulation of electrons (low e-flux) on FeMo-co in the presence of other (photo)chemical donors (811) could allow less reduced FeMo-co states (e.g., E2) to oxidize by evolving H2 before N2 binds. It is also possible that the binding of the CdS nanorod to the MoFe protein could induce protein conformational changes necessary to achieve N2 reduction that normally occur upon Fe protein binding (9).

The ability to create complexes between nanomaterials and MoFe protein and other enzymes establishes that photoexcited electrons can be used to drive difficult catalytic transformations while providing new tools for mechanistic investigations. Likewise, the light-harvesting properties of nanomaterials are highly tunable, and their unique optical properties can be used to probe in fine detail how changes in structure and energetics control electron transfer and macroscopic reaction rates. Biohybrid complexes can be used to examine how the flux and thermodynamics of photoexcited electron transfer influence the turnover and fidelity of catalytic product formation. Pairing biohybrid photochemical complexes with time-resolved methods is likely to enable profound new insights into the stepwise processes that underpin these challenging chemical reactions.

Supplementary Materials

www.sciencemag.org/content/352/6284/448/suppl/DC1

Materials and Methods

Figs. S1 to S3

Tables S1 to S13

References (2328)

References and Notes

  1. Materials and methods are available on Science Online.
  2. Acknowledgments: K.A.B. and P.W.K. were supported by a Laboratory Directed Research and Development Program seed project at the National Renewable Energy Laboratory for CdS:MoFe protein photochemical H2 production experiments, and by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences; and the U.S. Department of Energy under Contract no. DE-AC36-08-GO28308 with the National Renewable Energy Laboratory for CdS:MoFe protein biohybrid N2 reduction experiments. M.B.W., H.H., and G.D. conducted nanocrystal synthesis and ligand exchange under support by U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering under Award DE-SC0010334. D.F.H., A.R., N.K., S.K., L.C.S., and J.W.P. were supported for nitrogenase purification and product quantification as part of the Biological and Electron Transfer and Catalysis (BETCy) EFRC, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science (DE-SC0012518). The authors thank W. Tumas and R. Greene for thoughtful advice and many helpful discussions, and B. Hoffman for helpful discussions and constructive reading of the manuscript. Data are available in the supplementary materials. K.A.B. preformed photochemical experiments, including colorimetric NH3 measurements; N.K., A.R., D.F.H., and S.K. preformed nitrogenase and MoFe protein purifications, physiological nitrogenase assays, and fluorometric NH3 measurements; M.B.W. and H.H. performed CdS nanocrystal synthesis, ligand exchange, and transmission electron microscopy imaging; K.A.B, L.C.S., G.D., J.W.P., and P.W.K. conceived and designed the study. All authors contributed to the writing of the manuscript.
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