Graphitic carbon nitride supported Fe single-atom nanozymes synergize with nitrate reductase for photobiocatalytic nitrate conversion

doi:10.21203/rs.3.rs-3756664/v1

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Graphitic carbon nitride supported Fe single-atom nanozymes synergize with nitrate reductase for photobiocatalytic nitrate conversion

https://doi.org/10.21203/rs.3.rs-3756664/v1

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Nitrate reduction is of paramount importance for both the restoration of natural ecosystems and the sustainable development of chemical industries, but faces challenges in the rate-limiting step of nitrate-nitrite conversion and the poor product selectivity of the multielectron reactions. Herein, we demonstrate a nitrate-to-nitrite photosynthesis route catalyzed by a biotic-abiotic nanoarchitecture composed of carbon nitride (C₃N₄) supported Fe single-atom nanozymes combined with native nitrate reductases. Under visible light irradiation, the photobiocatalyst exhibits a state-of-the-art capability for nitrate conversion, with nearly 100% selectivity of nitrate and an unprecedented reaction kinetic constant that far exceeds those of all reported visible-light-driven photocatalysts. The strong binding affinity of nanozyme to the reaction substrate contributes to the 68.9- and 20.2-fold enhancement in the activity compared to artificial C₃N₄ photocatalyst and natural NarGHI biocatalyst, respectively, far exceeding the biological enzymes and reported enzyme-mimicking nanomaterials for nitrate conversion. Mechanistic studies at the atomic- and molecular levels reveal that Fe single atoms and cyano (-C ≡ N) groups on C₃N₄ mimic the heme b_D and heme b_P in the NarI subunit and construct a unique electron transfer chain between the biotic-abiotic interface under visible light irradiation, contributing to efficient and selective nitrate reduction to nitrite via photobiocatalysis. This work represents a promising biotic-abiotic platform to address the bottleneck problems of nitrate conversion, offering new opportunities for the sustainable removal of nitrogen from water and ammonia energy production with low energy consumption and carbon emission.

Physical sciences/Materials science/Materials for energy and catalysis/Photocatalysis

Physical sciences/Chemistry/Catalysis/Photocatalysis

Physical sciences/Chemistry/Photochemistry/Photocatalysis

Nitrate is the most stable nitrogen compound and is widely distributed in nature. The conversion of nitrate is a key process in both the natural ecosystems and the chemical industry¹. Environmentalists are pursuing the sustainable removal of nitrogen from the water via low-carbon approaches, such as the combined process of partial denitrification and anaerobic ammonium oxidation (PD-ANAMMOX), to maintain the global nitrogen balance². The targeted production of valuable ammonia via nitrate reduction reaction (NO₃^-RR) is also being actively explored in renewable energy research, representing an appealing and supplementary alternative to the energy-consuming Haber-Bosch process^3–5. Despite significant advances in the artificial conversion of nitrate over the past decades, both the efficiencies of the PD-ANAMMOX process and ammonia synthesis reaction are restricted by the poor selectivity and sluggish reaction kinetics for the initial conversion of nitrate into nitrite, which has been determined to be the rate-limiting step in the entire reaction cycle^6,7. With a wide range of oxidation states (-3 to + 5), the precise manipulation of nitrogen redox states toward the target compound is so far technically difficult.

In nature, living microorganisms can trigger a vast variety of complex chemical reactions with high selectivity and low substrate activation barriers by transferring multiple electrons between the catalytic centers of redox enzymes and reaction substrates^8–10. Numerous studies have attempted to mimic the functions of these microorganisms using synthetic materials, while the construction of biotic-abiotic nanoarchitectures provides a facile way to overcome the complexity of biological systems. Especially, there has been increasing concern about developing semi-artificial photosynthetic systems, leading to the synergistic conversion of CO₂, H₂O, and N₂ into value-added products with renewable solar energy^11–15. However, due to the rapid chain reaction from nitrate to dinitrogen in denitrifying bacteria, the construction of whole-cell-based architectures for selectively converting nitrate into nitrite remains a huge challenge. Furthermore, the bioconversion of nitrate relies on the respiratory chain terminated by a membrane-bound quinol-nitrate oxidoreductase, NarGHI¹⁶. By now, it is still notoriously difficult to construct the entire electron transfer pathway by mimicking the function of NarI, an essential transmembrane subunit that bridges the exogenous reaction substrate and the catalytic dimer subunit (NarGH) via two low-span hemes.

Atomically dispersed metal sites provide a promising platform for mimicking the well-defined geometric and electronic structures of the active centers in the metalloenzyme subunits. By combining the advantages of enzymatic, homogeneous, and heterogeneous catalysis, several types of single-atom mimetic enzymes have been developed and demonstrated comparable catalytic activity and kinetics to natural enzymes^17–21. This inspires us to develop a single-atom-based system that can efficiently and directionally convert nitrate into nitrite, to address the aforementioned problems of nitrate conversion reactions. We demonstrate that Fe single atoms and -C ≡ N groups on graphitic C₃N₄ with unique geometric and electronic structures could mimic the low-span hemes in the NarI subunit, and thus prepared the Fe/C₃N₄-CN (CN is used to represent -C ≡ N groups) nanozyme with charge transferring capability for catalyzing multi-electron reactions. As shown in Fig. 1, the integration of Fe/C₃N₄-CN with natural NarGH dimer in nitrate reductase (Fe/C₃N₄-CN/NarGH) forms the highly reactive catalytic centers for nitrate conversion. This semi-artificial nanoarchitecture can photosynthesize nitrite with nearly 100% selectivity, together with a striking reaction kinetic constant (0.0164 min^− 1) that is 20.2 and 68.9 times higher than biocatalysis and photocatalysis, respectively. Intermolecular interaction analysis, photophysical characterizations, and theoretical computation suggest that the unprecedented performance of the constructed nano/natural Fe/C₃N₄-CN/NarGH hybrids is ascribed to the efficient transfer of photogenerated electrons from the single-atom nanozymes to nitrate reductase.

Theoretical predictions

In the heterotrimeric NarGHI, the NarI subunit mediates the electron transfer from the quinol substrate to the [3Fe-4S] cluster FS4 in NarH and then the Mo-bisMGD cofactor in NarG¹⁶. Thus, mimicking the functionalities of the oxidation sites (b_D) and reduction sites (b_P) in NarI is crucial for constructing the artificial electron transfer chain. Inspired by the specific configuration of nitrate reductase, we hypothesized that introducing spatially separated Fe single atoms and -C ≡ N groups to C₃N₄ could simulate the dual-active sites in the NarI subunit for photosynthesis. To understand how the combined nanozyme interacts with NarGH, we performed molecular dynamics (MD) simulations to investigate their binding behaviors at the molecular level. According to the positions of FeS clusters in NarH, four initial conformations with FS1, FS2, FS3, and FS4 clusters approaching the surface -C ≡ N groups of C₃N₄ were constructed (conformation I-IV), respectively (Supplementary Fig. 1, 2). After 100 ns simulation, all conformations can be absorbed onto the edge surface of dual-site C₃N₄ (Supplementary Fig. 3 and Fig. 2a). Conformation IV exhibits the strongest interaction energy (-4900 kJ mol^-1) (Fig. 2b) between NarGH and nanozyme, and thus is determined to be the most stable binding conformation with residues anchored into the layers of Fe/C₃N₄-CN (Supplementary Table 1). This is also consistent with the slight variations in the root-mean-square deviation (RMSD) and radius of gyration (Rg) of NarGH (Supplementary Fig. 4), revealing an ignorable change in the structural conformation of NarGH after adsorbed on nanozymes²².

Density functional theory (DFT) calculations were further performed to evaluate the contribution of the isolated Fe atoms in C₃N₄ to the redox reactions. A Fe-N₄ configuration with an average bond distance of 1.96 Å was first constructed (Supplementary Fig. 5) to mimic the oxidative heme b_D in NarI for the dehydrogenation of duroquinol (DQH₂), a molecular electron donor that is commonly used to simulate the quinol substrate in vivo²³. Figure 2c shows the calculated Gibbs free energies of reactions on the biological heme b_D, pristine bulk C₃N₄ (B-C₃N₄), and artificial nanozymes via the reaction pathways of proton-coupled electron transfer (PCET). Among the three models, the Fe-N₄ site in Fe/C₃N₄-CN has the strongest adsorption energy of -0.76 eV for DQH₂, followed by B-C₃N₄ and heme b_D with adsorption energies of -0.60 eV and − 0.37 eV, respectively, indicating that Fe/C₃N₄-CN nanozyme has greater binding affinity for DQH₂. Moreover, more energy was released from the cleavage of the O-H bond in DQH₂ at the Fe-N₄ site, manifesting the superiority of our protocol for promoting the exothermic dissociation of the first proton by mimicking the NarI subunit. This can be further confirmed by the variation in the charge density distribution. Compared with heme b_D and B-C₃N₄, a noticeable charge redistribution is observed for the Fe/C₃N₄-CN nanozyme when coordinated with DQH (Fig. 2d), suggesting the strong interfacial interactions between Fe/C₃N₄-CN and DQH intermediate, which facilitates the electron transfer from DQH to the Fe-N₄ sites of Fe/C₃N₄-CN.

To verify the feasibility of mimicking the reductive heme b_P, we then calculated the site-site radial distribution functions (RDF) of Fe core active sites in the FeS clusters of NarH and -C ≡ N groups of Fe/C₃N₄-CN²⁴ (Fig. 2e). A prominent peak in the g (r) function at the distance of approximately 7 Å is observed for conformation IV, within the range of theoretical distance required for the direct edge-to-edge physiological electron transfer (14 Å)²⁵, whereas the other peaks locate out of 30 Å, indicating an electron transfer path from nanozymes to biological enzymes through FS4. Therefore, both thermodynamic and kinetics analyses predicted that the single-atom nanozymes could efficiently acquire and inject electrons to NarGH enzymes for the synergistic conversion of nitrate.

Synthesis and structural characterizations of Fe/C₃N₄-CN nanozyme

The synthesis of Fe/C₃N₄-CN nanozyme is schematically illustrated in Fig. 3a. By using NaCl crystals as a nucleation inhibitor, Fe was atomically dispersed in the precursor framework by rapid freezing with liquid nitrogen²⁶. The subsequent pyrolysis of precursors in the confined space of the molten salts induced the incomplete condensation of the tri-s-triazine units, forming -C ≡ N moiety on the edge of Fe/C₃N₄-CN. X-ray powder diffraction (XRD) analysis (Supplementary Fig. 6) reveals a typical graphitic phase structure of Fe/C₃N₄-CN. Transmission electron microscopy (TEM) observations suggest the formation of small nanoparticles (NPs) with a narrow size distribution (Supplementary Fig. 7, Supplementary Fig. 8a), which is different from those of B-C₃N₄ and C₃N₄ nanosheets without Fe single atoms (C₃N₄-CN) (Supplementary Fig. 8b, 8c). The elemental mapping results show that C, N, and Fe elements are homogeneously distributed in Fe/C₃N₄-CN NPs (Fig. 3b), which is further confirmed by the high-angle-annular-dark-field scanning transmission electron microscopy (HAADF-STEM). In Fig. 3c, the appearance of isolated bright dots (circled in red) proves the atomic dispersion of Fe on the C₃N₄ matrix. The intensity profiles along lines 1 and 2 suggest that these bright dots are individual Fe atoms, or two isolated Fe atoms (Fig. 3d). Based on the inductively coupled plasma mass spectrometry (ICP-MS) results, the optimal loading mass of Fe is about 0.25 wt%, approaching the content of Fe in the hemes of biological NarI subunit (0.53 wt%).

The coordination structure of Fe active sites in Fe/C₃N₄-CN was next probed by X-ray absorption fine structure (XAFS) spectroscopy. For the Fe K-edge X-ray absorption near edge structure (XANES), the absorption edge of Fe/C₃N₄-CN is located between those of FeO and Fe₂O₃ (Fig. 3e) revealing a valance state of Fe between + 2 and + 3. Besides, the appearance of additional peaks at 709.6 and 722.8 eV in the Fe 2p X-ray photoelectron spectroscopy (XPS) result implies the coordination of Fe with nitrogen (Supplementary Fig. 9)²⁷. Fourier transformed (FT) k³-weighted extended-XAFS (EXAFS) spectrum of Fe/C₃N₄-CN displays only one peak at 1.58 Å (Fig. 3f), corresponding to the Fe-N bonds in iron-porphyrin (Fe-Pc), in consistent with the XPS result. No peak corresponding to the Fe-Fe (2.2 Å) bond is detected, indicating the atomic dispersity of Fe sites²⁸. This can be further validated by the wavelet transform (WT) results, as only one intensity maximum at 1.4 Å assigned to the first scattering path of Fe-N is observed, differing from the Fe-Fe signal at around 2.1 Å in the spectrum of Fe foil²⁹ (Fig. 3g). According to the EXAFS fitting results (Supplementary Fig. 10), the coordination number of Fe in Fe/C₃N₄-CN is 3.9 (Supplementary Table 2), meaning that the isolated Fe is fourfold coordinated by N atoms, with an average Fe-N coordination distance of 1.99 Å, consistent with the DFT results.

The formation of -C ≡ N groups in Fe/C₃N₄-CN was validated by the emergence of the vibration absorption peak at 2170 cm^-1 in the Fourier transform infrared spectrum (FTIR, Fig. 3h), together with the new C 1s component at 286 eV in the XPS spectrum^30,31 (Fig. 3i). Solid-state ¹³C magic angle spinning (MAS) nuclear magnetic resonance (NMR) was performed to determine the formation location of -C ≡ N groups (Fig. 3j). Besides the intensive C_3N (1) and C_2N-NHx (2) peaks at the chemical shifts of 156.4 and 164.5 ppm, two additional peaks corresponding to carbon atoms in the -C ≡ N groups (3) and the neighboring carbon atoms (4) are distinctly observed at 121.3 and 170.8 ppm in the spectrum of Fe/C₃N₄-CN³². Therefore, we can conclude that these -C ≡ N groups are located at the apex of the melon structure (inset of Fig. 3j). With electron-withdrawing capability³³, these -C ≡ N groups pull the electron cloud of Fe atoms toward the aromatic rings, increasing the oxidative capability of the mimetic b_D sites, as evidenced by the increased electron density of pyridinic N in the high-resolution N 1s XPS spectrum of Fe/C₃N₄-CN (Supplementary Fig. 11).

Performance characterization of Fe/C₃N₄-CN/NarGH for nitrate conversion

Our Fe/C₃N₄-CN nanozymes can easily bind with the natural NarGH protein, because of their comparable particle sizes, solubilities, and molecular weights (Supplementary Fig. 7, 12, 13, and Supplementary Table 3)^13,16. We extracted the heterotrimeric NarGHI by purifying the crude enzyme from Paracoccus denitrificans via anion exchange followed by size exclusion chromatography and then obtained the NarGH dimer by heating NarGHI at 60 ºC to separate the loosely bounded NarI subunit^34,35 (Supplementary Fig. 14). The obtained NarGH bio-enzymes were directly hybridized with Fe/C₃N₄-CN nanozymes via electrostatic self-assembly under the neutral pH (Supplementary Fig. 15). Surface plasmon resonance (SPR) fitting reveals the fast binding kinetics between NarGH and Fe/C₃N₄-CN, with a high association rate (k_a = 9.60 × 10⁵ M^-1 s^-1) and a low dissociation rate (k_d = 7.05 × 10^− 5 s^-1) (Supplementary Fig. 16a). The steady-state affinity analysis confirmed the strong binding process, contributing to an affinity constant K_D of 16.7 nM for the assembled biohybrids³⁶ (Supplementary Fig. 16b), in agreement with the MD simulation. Confocal laser scanning microscopy (CLSM) further confirmed the tight connection between NarGH and Fe/C₃N₄-CN (Fig. 4a). Additionally, no obvious variation in the activity and secondary structure of NarGH was detected in the presence of Fe/C₃N₄-CN and light irradiation (Supplementary Fig. 17), indicating the good structural and conformational stability of NarGH, which is consistent with the low RMSD and Rg in the MD calculations.

A series of photo-enzymatic experiments were carried out under anaerobic conditions to evaluate the performance of Fe/C₃N₄-CN/NarGH nanoarchitectures for nitrate conversion, using DQH₂ as a physiological reductant. As shown in Supplementary Fig. 18a, the blank B-C₃N₄ or Fe/C₃N₄-CN NPs without NarGH could not catalyze nitrate reduction. B-C₃N₄ without -C ≡ N groups and Fe active sites could only slightly improve the catalytic efficiency of the NarGH enzyme. In contrast, the integration of Fe/C₃N₄-CN with NarGH yields an unprecedented activity for converting nitrate into nitrite with nearly 100% selectivity (Fig. 4b and Supplementary Fig. 18b). The calculated reaction kinetic constant (k) is up to 1.64 × 10^− 2 min^-1 (Supplementary Fig. 18c), which is 68.9 times higher than that of the artificial photocatalysis with B-C₃N₄ (2.38 × 10^− 4 min^-1) and even 20.2 times higher than that of the natural NarGHI enzyme (8.13 × 10^− 4 min^-1), contributing to a record activity comparison to all reported visible-light-driven catalysts for nitrate conversion (Supplementary Table 4). It should be mentioned that no NO₂^- was generated when inactivated enzymes were used or when no light or Fe/C₃N₄-CN was used (Supplementary Fig. 18d), revealing the critical role of interfacial electron transfer between the Fe/C₃N₄-CN and NarGH enzyme for nitrate conversion.

We then compared the reaction kinetics of biological NarGHI (Fig. 4c) with that of the Fe/C₃N₄-CN/NarGH biohybrids (Fig. 4d) by changing the concentration of DQH₂ substrate while keeping the other parameters constant³⁷. Both steady-state kinetic curves follow the typical Michaelis-Menten model in dependence on the concentration of DQH₂. By fitting the double reciprocal (Lineweaver-Burk) plots (inset), we gain the maximum initial velocity (V_max) and Michaelis-Menten constant (K_m), which represent the catalytic efficiency and binding affinity, respectively. To better demonstrate the kinetics based on Fe active sites, we further calculated the parameters of k_cat and k_cat/K_m, which are displayed in Supplementary Table 5. The K_m value of Fe/C₃N₄-CN/NarGH (0.09 mM) is much lower than that of NarGHI (0.65 mM), indicating the higher binding affinity of Fe/C₃N₄-CN/NarGH to the quinol-type electron donors, which is well consistent with the adsorption energy results in the DFT calculations³⁸. Accordingly, Fe/C₃N₄-CN/NarGH catalyzes DQH₂ oxidation with 8.3 times higher efficiency (k_cat/K_m = 4.31 × 10³ M^-1 min^-1) compared to NarGHI (k_cat/K_m =5.23 × 10² M^-1 min^-1). The same conclusion was obtained by calculating the reaction kinetics as a function of nitrate concentration (Fig. 4e, f), with K_m reduced from 0.26 to 0.13 mM and k_cat/K_m increased from 8.61×10² to 3.48×10³ M^-1 min^-1. The maximum initial velocity (V_max) of 23.7 µmol NO₃^- mg_catalyst^-1 min^-1 is even several orders of magnitude higher than the reported nitrate reductase and nanozymes (Supplementary Table 6), demonstrating the great potential of our nanozyme/bioenzyme hybrids for overcoming the kinetic barrier of the rate-limiting reaction during nitrate reduction.

It is worth noting that Fe single-atom nanozymes do not suffer from the cost limitations of natural enzymes. Only expensive DOH₂ can be used for NarGHI due to the substrate specificity of the natural NarI subunit (Fig. 4g and Supplementary Fig. 19, 20, Table 7). However, several universal reagents, such as TEOA, glycerol, and ascorbic acid, can be employed as electron donor for our nanozymes with comparable activity to that of DQH₂. (Fig. 4h and Supplementary Fig. 21). In addition, confirmations of the two heme mimetic centers in Fe/C₃N₄-CN are able to remain stable under reaction conditions. Hence, 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), a common inhibitor for biological NarI¹⁶, presented a slight impact on the catalytic activity of Fe/C₃N₄-CN/NarGH nanoarchitectures (Supplementary Fig. 22). As demonstrated in Supplementary Fig. 23, the used photobiocatalysts can be easily recycled by increasing the ionic strength of the reaction solution. Therefore, the Fe/C₃N₄-CN/NarGH nanoarchitectures can provide a low-cost and durable catalytic system for the continuous conversion of nitrate into nitrite.

Mechanistic studies of the electron transfer across the abiotic-biotic interface of Fe/C₃N₄-CN/NarGH

The role of enzyme-mimicking sites on Fe/C₃N₄-CN in the photo-enzyme synergistic reaction was finally fundamentally investigated. DFT calculations first predicate that Fe single atoms and -C ≡ N groups create three empty bands composed of impurity energy levels between the conduction band (CB) and valence band (VB) of Fe/C₃N₄-CN (Supplementary Fig. 24). This induces a redshift of the absorption edge toward the visible region (Supplementary Fig. 25a and b), together with an elevation of the CB level position by 0.19 eV (Supplementary Fig. 25c and d), which should be favorable for the nitrate photoreduction³⁹. Furthermore, combined with the electron paramagnetic resonance (EPR) analysis (Supplementary Fig. 26), we disclose a unique process of electron cloud redistribution. This can form an electron-rich center around -C ≡ N groups on the aromatic rings and an electron-deficient center around Fe single atoms, which is also evidenced by the right shift of C = N-C peak in the XPS results (Supplementary Fig. 11 and Fig. 3i). Such spatial separation of charge carriers can notably improve the photoactivity of Fe/C₃N₄-CN, as evidenced by its distinctly quenched photoluminescence (PL), shortened PL lifetime, and increased photocurrent response (Supplementary Fig. 27, Table 8, and Fig. 28).

We next monitored the charge transfer behaviors in the Fe/C₃N₄-CN/ NarGH nanoarchitectures. Hybridization with NarGH enzymes causes a remarkable fluorescence quenching of Fe/C₃N₄-CN (Supplementary Fig. 29), together with a decrease in the average PL lifetime from 1.82 to 1.04 ns (Fig. 5a, Supplementary Table 9). In contrast, no variation in the lifetime decay is detected for the hybrids of biological NarGH and Fe/C₃N₄ without -C ≡ N groups. This demonstrates the critical role of -C ≡ N groups in transferring photoinduced electrons from Fe/C₃N₄-CN to NarGH, resulting in the enhancement in the transient photocurrent response (Supplementary Fig. 30).

The charge dynamics of the excited Fe/C₃N₄-CN/NarGH biohybrids were probed by transient absorption spectroscopy (TAS). In the spectrum of Fe/C₃N₄-CN, a distinct absorption peak appears at ~ 678 nm (Supplementary Fig. 31). This positive absorption is associated with photogenerated electrons, since AgNO₃ and TEOA as electron and hole scavengers attenuate and enhance the signal intensity, respectively⁴⁰ (Supplementary Fig. 32). The addition of NarGH significantly reduces the absorption signal of Fe/C₃N₄-CN, suggesting the occurrence of electron transfer from Fe/C₃N₄-CN to NarGH. We thereafter monitored the decay kinetics of photoelectrons by the time profiles of absorption probed at 678 nm. All curves follow a biexponential decay, with a fast component τ₁ and another slow component τ₂ that can be ascribed to electron trapping by surface states near the CB and the exciton recombination, respectively (Supplementary Table 10)²⁸. Both lifespans show a distinct decrease (Fig. 5b), which can not be observed for the NarGH/B-C₃N₄ hybrids (Supplementary Fig. 33). This well confirmed the efficient electron transfer from Fe/C₃N₄-CN to the FS4 centers in NarGH subunits⁴¹. We finally measured the apparent quantum yield (AQY) of Fe/C₃N₄-CN/NarGH biohybrids for nitrate reduction under various monochromatic light conditions. In Fig. 5c, the AQY value decreases with increasing light wavelength, and it qualitatively tracks the characteristic absorption of Fe/C₃N₄-CN. At 400 nm, the synergistic system shows a peak AQY of 24%, which is 8.9 times higher than that of photocatalysis. Under visible-light irradiation at 550 nm, the value still reaches 1.8%, validating the great potential of our nanozyme/bioenzyme hybrids for the efficient photoconversion of nitrate.

Application of the photobiocatalytic system

We first evaluated the applicability of the Fe/C₃N₄-CN/NarGH hybrids in the environmental field for the sustainable removal of nitrogen from water. Photobiocatalytic reduction of nitrate can be employed as a partial denitrification process to accumulate nitrite substrate for the anammox reactions, which has been considered a promising option to energy-neutral wastewater treatment but suffers from the inadequate supply of nitrite as an electron accepter^2,42. It is clear from Fig. 5d and Supplementary Fig. 34 that Fe/C₃N₄-CN/NarGH reduces nitrate more efficiently than denitrifying bacteria, ensuring a high concentration of nitrite substrate for the subsequent removal of nitrogen with anammox bacteria. Compared to the biological pathway via denitrifying and anammox bacteria, the combination of photobiocatalysis and anammox bacteria increases the removal efficiency of total nitrogen (TN) from 25.5 ± 0.7% to 94.2 ± 0.6%. The efficient and selective conversion of nitrate into nitrite also contributes to the artificial synthesis of ammonia, which has been considered a zero-carbon molecule that can provide the required energy storage medium for renewable sources⁴³. The photobiocatalytic system produces nitrite with low energy input, thus overcoming the energy barrier for the rate-limiting step of nitrate-to-nitrite conversion during NO₃^-RR. To prove this, we reduced ¹⁴NO₃^- by Fe/C₃N₄-CN/NarGH photobiocatalysts and mixed the ¹⁴NO₂^- product with isotopically labeled ¹⁵NO₃^- to perform the electrocatalytic production of NH₃ over Cu, Co, and Ni foam electrodes, respectively^44–46. As can be seen from Fig. 5e, at the optimal potentials (Supplementary Fig. 35), more than 95% of NH₃ products (dissolved as NH₄⁺) are derived from the reduction of ¹⁴NO₂^- (Fig. 5f), implying that the photobiocatalytically generated nitrite can be more easily converted into green NH₃ energy via subsequent catalysis with low reaction barrier (Supplementary Fig. 36). Overall, the Fe/C₃N₄-CN/NarGH photobiocatalysts exhibit great application potentials for the conversion and utilization of nitrate with low energy consumption and carbon emission.

In summary, we demonstrate an artificial photosynthetic system composed of Fe single-atom nanozymes and native nitrate reductases for the effective and selective conversion of nitrate to nitrite. Advanced characterizations and theoretical calculations revealed that Fe single atoms and -C ≡ N groups on carbon nitride polymer precisely mimic the well-defined electronic structures of the redox centers in biological NarI subunits, constructing the artificial pathway for electron transfer from Fe/C₃N₄-CN to the NarGH subunits. Under visible light illumination, the biotic-abiotic nanoarchitecture exhibits a state-of-the-art capability for nitrate conversion, with a V_max of 23.7 µmol NO₃^- mg_catalyst^-1 min^-1 that is several orders of magnitude higher than the biological enzymes and reported enzyme-mimicking nanomaterials. The transient spectra analysis confirmed that the extraordinary photobiocatalytic activity of Fe/C₃N₄-CN/NarGH hybrids originated from the efficient charge transfer between Fe/C₃N₄-CN and nitrate reductase. The photobiocatalytic conversion of nitrate to nitrite breaks the rate bottleneck of nitrate reduction reactions, thus significantly contributing to the energy-neutral wastewater treatment via anammox reactions and artificial synthesis of ammonia with low energy consumption and carbon emission. This study not only provides atomic- and molecular-level insights into the biotic-abiotic interactions, but also offers a new avenue for developing artificial photobiocatalytic systems for environmental and energy applications.

All the reagents were analytical grade and purchased from Sigma-Aldrich and China Chemical Reagent without further purification. All ultrapure water was obtained from a Millipore Millipak Express 40 system.

Synthesis of single-atom nanozyme. For the synthesis of Fe/C₃N₄-CN, 4 g of dicyandiamide was dissolved into 100 mL of ultrapure water to form a homogenous solution. 10 g of NaCl and 17 mg of iron (II) acetate were then added to the above solution with vigorous stirring at room temperature. The solution was quickly frozen in liquid nitrogen and dried in a freeze-dryer to achieve the precursor powders, which were pyrolyzed at 550°C for 4 h in a tube furnace under an N₂ atmosphere. Fe/C₃N₄-CN was finally obtained after washing and drying treatment. For comparison, Fe/C₃N₄ and C₃N₄-CN were prepared by the same method, except for the addition of NaCl or iron (II) acetate. B-C₃N₄ was synthesized by the pyrolysis of dicyandiamide at 550°C for 4 h in a tube furnace under the N₂ atmosphere.

Characterizations. X-ray diffraction (XRD) measurement was carried out using Bruker D8 equipment with Cu Kα radiation. Transmission electron microscopy (TEM, JEM-2100F) was used to observe the morphologies of samples. Atomic resolution imaging was collected by a high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM, JEOL-ARM-300) with an accelerating voltage of 300 kV. X-ray absorption spectra (XAS) were collected at the 1W1B station in the Beijing Synchrotron Radiation Facility (BSRF). X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) were acquired in the fluorescence mode at room temperature, with energy calibrated using Fe foil. X-ray photoelectron spectroscopy (XPS) was measured on an ESCALAB 250Xi XPS System (Thermo Fisher Scientific, UK). Electron paramagnetic resonance (EPR) measurements were carried out using a Bruker A300 spectrometer. Fourier transform infrared spectra (FTIR) were collected by a Nicolet iN10MX spectrometer (Thermo Fisher Scientific). Solid-state ¹³C magic angle spinning nuclear magnetic resonance (MAS-NMR) spectra were measured on a Bruker AVANCE III 400 MHz WB spectrometer at room temperature. Photoluminescence (PL) spectra were collected by a HITACHI F-7000 spectrophotometer with an excitation wavelength of 369 nm. The kinetics of fluorescence was investigated by a time-resolved fluorescence spectrometer (Edinburgh, FLS-920), with excitation by the ultrafast nanosecond laser (nF980) at 369 nm. The diffuse reflectance spectroscopy (DRS) of different catalysts was detected using a HITACHI U-3900 spectrophotometer with BaSO₄ as a reference. An inductively coupled plasma atomic emission spectrometry (ICP-AES, Agilent 710-ES) was employed to determine the mass loading of Fe in Fe/C₃N₄-CN.

Bacteria growth and crude enzyme extraction. The nitrate reductase was expressed and purified from P. denitrificans (ATCC 19367) according to the previous reports³⁵. Typically, P. denitrificans were cultured anaerobically in the 1 L batch at 37 ºC with acetate as a carbon source and nitrate as a terminal electron acceptor to induce the NarGHI expression. The culture medium, containing 5 g KNO₃, 6 g CH₃COONa, 1 g NH₄Cl, 2.4 g KH₂PO₄, 11.7 g Na₂HPO₄•12H₂O, 6.6 mg Na₂-EDTA, 2.4 mg FeCl₃•6H₂O, 20 µg MnCl₂•4H₂O, 0.2 mg Na₂MoO₄•2H₂O, 0.1 mg CuCl₂•2H₂O, and 0.3 mg ZnCl₂, was sterilized under 121 ºC for 15 min. After cooling down, 100 mg of MgSO₄•7H₂O was added to the medium through a sterilized filter. The P. denitrificans cells were harvested when the OD₆₀₀ reached 2.0 after inoculation for about 36 hours. The cell pellet was washed with 20 mM PBS (pH 7.4) three times, then resuspended in Buffer A (20 mM HEPES, pH 7.4, 0.1 mM dithiothreitol, 0.5 mM EDTA, 1 mM cysteine, 0.1% (v/v) Nonidet P-40). The cells were lysed by probe ultrasonication (200 W, cycle of 2 s operation followed by 8 s stand by) for 8 min at 4°C, and the membrane vesicles were solubilized by Nonidet P-40 to give a final reagent ratio of 2% (v/v) with constant stirring at 4°C for 2 hours. The soluble crude enzyme was achieved after removing the cell debris and insoluble protein by centrifugation (57000 g, 30 min, 4°C).

Purification of nitrate reductase. The biological NarGHI was purified via the steps of ammonium sulfate precipitation and chromatographic separation. In brief, solid ammonium sulfate (36.2 g) was slowly added into the solution of the crude enzyme, which was brought to 35% saturation of ammonium sulfate with a final volume of 200 mL. The mixture was stirred at 4°C for 2 hours and then settled overnight. The precipitate was collected by centrifugation at 14000 g for 30 min. The precipitate was redissolved in Buffer A and applied to a Source 15Q 10/100 GL column (GE-Healthcare) which had been equilibrated with 30 ml Buffer A. The bound fractions were eluted with a gradient of 0-300 mM NaCl in Buffer A. After that, the active fractions were concentrated by concentrating vials (50 kDa, Millipore Merck, Ltd) and further purified by an exclusion chromatography equipped with a Superdex 200 Increase 10/300 GL column (GE-Healthcare) in Buffer B (20 mM HEPES, pH = 7.4, 0.1 mM dithiothreitol, 0.5 mM EDTA, 0.1% (v/v) Nonidet P-40, 5% glycerol). All the chromatographic steps were operated on an AKTA purifier system (GE-Healthcare) at 4°C. The NarGH dimer was obtained after the partial dissociation of the NarGHI complex by incubating in 1% n-Octyl-D-glucopyranoside (OG) at an elevated temperature (60°C). The purity of nitrate reductase was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the concentration of protein was measured by a Bradford assay with BSA as a standard. Before use, the aliquot substrate was flash-frozen in liquid nitrogen and subsequently stored at -80°C.

Activity assay of nitrate reductase. The activity of nitrate reduction was evaluated by sodium hydrosulfite (Na₂S₂O₄), using methyl viologen (MV) as an electron donor. Typically, 80 µL of 100 mM Na₂S₂O₄, 50 µL of 20 mM MV, 1.80 mL of 40 mM PBS (pH 7.4), 40 µL of 10 mM NaNO_3, and 30 µL of enzyme solution were successively added into a 2 mL tube, which was then incubated in an air bath shaker (150 rpm) at 37 ºC for 30 min. The activity of nitrate reductase was evaluated by measuring the concentration of nitrite. One unit (U) of enzyme activity is defined as reducing one nmol of nitrate per minute, and the activity unit is expressed as U mg^-1.

SPR analysis. SPR analysis was conducted to evaluate the kinetics and affinity of the interactions between Fe/C₃N₄-CN and NarGH dimer, which was performed on a Biacore 8k instrument (GE-Healthcare) with a CM5 sensor chip. The running buffer was 10 mM phosphate solution (PB, pH 6.5) containing 0.05% of Tween P20. According to the pH scouting, NarGH was immobilized on the sensor chip using an amine coupling kit from GE Healthcare (BR-1006-33) in a 10 mM sodium acetate (pH 4.0) solution. The binding of Fe/C₃N₄-CN and NarGH was operated by injecting five different concentrations of Fe/C₃N₄-CN (1.22, 2.44, 4.88, 9.76, and 19.52 nM) in triplicate over both flow cells to allow for the comparative analyses relative to the binding to the reference flow cell. The CM5 sensor chip was regenerated with 2 M MgCl₂ after each injection cycle. The binding curves were recorded by the double reference method to correct the instrument noise, bulk shift, and drift during sample injections. The obtained sensor curves were fitted by a 1:1 Langmuir binding model for the kinetic and affinity analysis using the BIACORE INSIGHT EVALUATION software.

Confocal laser scanning microscopy. Confocal laser scanning microscopy images were obtained using a Leica TCS SP8 II confocal imaging system equipped with a CFI Plan Apo Lambda 100× Oil objective. The biological NarGH was firstly stained with FITC, and the unbonded dyes were washed with PBS (20 mM, pH 7.2) using an ultrafiltration centrifuge tube (50 kDa, Millipore Merck, Ltd). The unstained Fe/C₃N₄-CN could emit bright blue fluorescence under light irradiation. The stained NarGH was incubated with the Fe/C₃N₄-CN solution in PBS (20 mM, pH 7.2), which was then dropped onto the glass slides for microscopic observation.

Light-driven nitrate conversion experiments. The photobiocatalytic conversion of nitrate was performed in a sealed system under Xe lamp (China Education Au-light) illumination (λ > 400 nm) with an intensity of 30 mW cm^-2. Specifically, the experiment was conducted in a 2 ml quartz reactor that containing 1 mM NO₃^-, 0.5 mg mL^-1 Fe/C₃N₄-CN nanozyme, 4 mM DQH₂, and 1.0 mg mL^-1 NarGH dimer in PBS (20 mM, pH 7.0). According to the molecular weight of Fe/C₃N₄-CN (93 kDa) and NarGH (193 kDa), equal volumes of Fe/C₃N₄-CN (2.5 mg mL^-1) and NarGH dimer (5.0 mg mL^-1) were mixed to obtain the biohybrids with a molar ratio of 1:1. To ensure the anaerobic condition, the sealed quartz reactor was vacuumed and purged with Ar gas at least three times for 20 minutes each time. During the photocatalytic reactions, 0.3 mL of solution samples were withdrawn periodically for analysis. After filtration through a 0.45 µm microporous filter membrane, the concentrations of NO₃^- and NO₂^- in the solution were determined by an ions chromatography (ICS-2000, DIONEX, USA). The inactivated NarGH in control experiments was obtained after being sterilized under 121 ºC for 15 min. We evaluated the catalytic activity and selectivity by calculating the kinetic constants for nitrate removal and nitrite generation, respectively. The efficiency of nitrite conversion was defined as: [produced NO₂⁻]/[reduced NO₃^-] × 100%. The AQY was calculated through the equation (Eq. 1),

$$\begin{gathered} {\text{AQY=}}\frac{{{\text{Number of reacted electrons}}}}{{{\text{Number of incident electrons}}}} \times 100\% \hfill \\ {\text{ =}}\frac{{(\upsilon \times {N_{\text{A}}} \times K)}}{{\frac{{I \times S \times \lambda }}{{h \times c}}}} \times 100\% \hfill \\ \end{gathered}$$

where υ is the reaction velocity of production of NO₂^- (mol s^-1), N_A is Avogadro's number (6.02 ×10²³ mol^-1), K is the reacted electrons per molecule (K = 2), h is Planck’s constant (6.62×10^–34 J s), c is the speed of light (3.0×10⁸ m s^-1), I is the light intensity (10 W m^-2), S is the area of irradiation (2.0 ×10^− 5 m²), λ is the light wavelength (nm). The incident wavelength was produced by a monochromator (7ISW151, SOFN INSTRUMENTS CO., Ltd), and the light intensity was determined by an optical power meter (PM100D, THORLABS).

Electrochemical analysis. For the preparation of the working electrode, 5 mg of Fe/C₃N₄-CN nanozyme was dispersed into 1 mL of 0.1 wt% Nafion solution with ultrasonication for 10 min. Then, 100 µL of the catalyst ink was loaded onto a piece of FTO glass and dried naturally. To prepare the biohybridized ink, the NarGH solution was mixed with the Fe/C₃N₄-CN solution with a volume ratio of 1:4. The transient photocurrent was measured by an electrochemical workstation (Gamry, Interface 1000) with a typical three-electrode system. FTO glass coated with catalysts, Ag/AgCl, and Pt foil was used as the working electrode, reference electrode, and counter electrode, respectively. 50 mM HEPES buffer solution containing 100 mM Na₂SO₄ was used as the electrolyte, which was degassed by purging with N₂ flow for 30 minutes. For the electrochemical synthesis of ammonia, the commercial Cu, Co, and Ni foams (1.0 × 1.0 cm²) were used as working electrodes, and 20 mM of PBS solution (pH 7.0) was used as electrolyte. 20 mM of ¹⁴N-labeled ¹⁴NO₃⁻ and ¹⁵N-labeled ¹⁵NO₃⁻ were used as nitrogen sources for electroreduction reactions, respectively. Isotopic competition experiments were carried out to identify the difficulty levels of nitrate and nitrite reduction. Specifically, 20 mM of ¹⁴NO₃⁻ was firstly reduced to ¹⁴NO₂⁻ via photobiocatalysis with Fe/C₃N₄-CN/NarGH biohybrids. The converted ¹⁴NO₂⁻ was then mixed with an equal amount of ¹⁵NO₃⁻ for the electrosynthesis of NH₃ over Cu, Co, and Ni electrodes, respectively. The reaction was carried out for 3.0 hours at each potential to determine the optimal potentials for different electrocatalysts. The amount of dissolved NH₃ in the solution was determined by a ¹H NMR spectrometer (JNM-ECA600, JEOL) with DMSO-d6 as the deuterium reagent and maleic acid as the internal standard.

Experiments of nitrogen removal with anammox bacteria. Bacterial denitrification and photobiocatalysis reactions were separately performed to accumulate NO₂^- for the subsequent removal of nitrogen from water with anammox bacteria. P. denitrificans, resuscitated and rejuvenated in Luria-Bertani (LB) medium with OD₆₀₀ of 1.6 ~ 1.8, were used for bacterial denitrification as previously described⁴⁷. Briefly, the cells harvested by centrifugation at 5000 rpm for 5 minutes were suspended in 20 mM PBS buffer (pH 7.4), which contained 2 mM NO₃^-, 2 mM CH₃COONa, and necessary trace elements. The anaerobic reaction was conducted at 30 ºC for 180 minutes to convert nitrate with P. denitrificans. For comparison, photobiocatalytic reduction of nitrate was employed as a partial denitrification process to generate nitrite substrate for anammox reactions. The mature aerobic granular sludge derived from an ongoing lab-scale reactor was used to afford the anammox bacteria. In a typical procedure, 2 g L^-1 of the anammox biomass was added into the above solutions produced by the denitrifying bacteria and photobiocatalytic reaction, respectively. 2 mM of NH₄Cl was used as nitrogen pollutant in water to be removed by anammox bacteria, and the converted nitrite in the above solutions was directly used as reaction substrate. To meet the metabolism of anammox bacteria, 0.18 mM KH₂PO₄, 2 mM KHCO₃, 0.95 mM CaCl₂•2H₂O, 0.81 mM MgSO₄•7H₂O, 0.094 mM Na₂CO₃, 0.018 mM FeSO₄•7H₂O, and 0.02 mM EDTA were further added⁴⁸. The nitrogen removal reactions were carried out at 37 ºC on a shaker with a rotational speed of 150 r min^-1, and the concentration of NH₃ was determined using Nessler's reagent.

The kinetics of biological NarGHI and Fe/C₃N₄-CN/NarGH biohybrid for nitrate conversion. The kinetic evaluation of the biological enzyme (NarGHI) and the nanozyme/bioenzyme hybrids was carried out in 4 mL of PBS (20 mM, pH 7.0) solution, using adequate DQH₂ as an electron donor and different concentrations of NaNO₃ as the substrate. Typically, 1.5 mL of enzyme solution containing NarGHI or NarGH, 0.25 mL of DQH₂ (30 mM), 1.85 mL of PBS, and various volumes of NaNO₃ solution were added into the sealed quartz vials, respectively. 2 mg of Fe/C₃N₄-CN catalysts were pre-dispersed into PBS for the hybridized group. After being purged with Ar gas at least three times, the nitrate reduction reactions were conducted at 37 ºC with or without light irradiations. A Michaelis-Menten curve was obtained by plotting the calculated V against the concentration of NaNO₃ or DQH₂ substrate. Finally, the Michaelis-Menten equation (Eq. 2) was fitted using GraphPad Prism version 8 software to determine the kinetic parameters.

V = V_max[S]/(K_m+[S]) (2)

where [S] is the concentration of substrates.

Femtosecond transient absorption spectra. The femtosecond pump-probe TAS measurements were conducted using a regenerative amplified Ti: sapphire laser system (800 nm, 70 fs, 60 nJ/pulse, and 1 kHz repetition rate). The laser light was split into two parts to generate the pump and the probe pulses. The first beam was used to generate 400 nm laser pulses by second harmonic generation through a splitting nonlinear optical crystal (α-BBO) which will act as the excitation pulses. The second path light was focused onto a sapphire or CaF₂ window to generate a continuum of white light, which was used as the probe light. The sample solution was placed in a 1 mm quartz cuvette, which was irradiated by the spatially aligned pump and probe beams. The detection beam was collimated and focused into a fiber-coupled spectrometer with a CMOS sensor to probe the differential optical density ΔOD = log₁₀[I_{0 sample}/ I_{ex sample} × I_{ex ref}/I_{0 ref}].

MD simulations. Atomic MD simulations were performed by the GROMACS (version 2020.6) simulation package⁴⁹. Amino acid sequences of NarG and NarH obtained from NCBI (https://www.ncbi.nlm.nih.gov/, Supplementary Table 11) were used to build the protein models via RoseTTAFold⁵⁰. These models were enriched with ligands and cofactors using AlphaFill⁵¹. The (100) plane at the edge of the C₃N₄ nanosheet was decorated with -C ≡ N for further simulations. The Amber99sb-ILDN force field⁵² and the General Amber force field (GAFF) were used to describe the proteins and the surface of heme molecules, respectively. The Universal force field (UFF)⁵³ was adopted for Fe atoms to cover the whole periodic table. Combining with the AFM result, the proteins were placed 5 Å above the C₃N₄ surface with a side length of around 120 Å and solvated with water molecules, as described by the TIP3P model. Sodium ions (Na⁺) or chlorine anions (Cl^-) were added as counterions to neutralize the charge of the system by randomly replacing water molecules. Semiisotropic NPT runs with fixed surface side lengths of 5 ns were performed after thousands of steps for energy minimization to equilibrate the system. Finally, production runs of 100 ns at 298 K were performed in the NPT ensemble. A cutoff length of 12 Å was implemented for the non-bonded interactions, and the Particle Mesh Ewald method⁵⁴ with a cutoff distance of 10 Å was applied for the long-range electrostatic interactions. All the covalent bonds involving hydrogen atoms were constrained using the LINCS algorithm⁵⁵. The Visual Molecular Dynamics (VMD) program was performed for structure visualization.

DFT calculations. The binding model of heme b_D for DQH₂ was constructed according to the structural environment of the pentachlorophenol (PCP) binding site (Supplementary Table 12) in the NarI subunit (PDB ID:1Y4Z)⁵⁶. All the calculations were performed in the framework of the density functional theory with the projector-augmented plane-wave method, as implemented in the Vienna Ab Initio Simulation Package (VSAP)⁵⁷. The projector augmented wave (PAW) potentials and the generalized gradient approximation (GGA) with the Perdew-Burke Ernzerh (PBE) formulation were used to describe the electron-ion interaction^58,59. The cut-off energy was set as 400 eV for the expansion of the plane wave. A vertical vacuum layer of 20 Å was added to avoid the artificial interactions between the periodic images. Due to the large size of a supercell, the K-space was sampled with a grid of 1×1×1 using the Monkhorst-Pack scheme³³. The convergence criteria for the energy self-consistent iteration and structure relaxation were 10^− 4 eV and 0.03 eV/Å, respectively. A D3 correction method was used to describe the van der Waals interactions for better accuracy. The adsorption energies (ΔG) were obtained by (Eq. 3)

ΔG = ΔEDFT + ΔZPE – TΔS (3)

where ΔEDFT is the reaction energy calculated from DFT; ΔZPE is the zero-point energy; ΔS is the change in entropy. For the DQH₂ molecule, the ΔS values were obtained from the standard database in the NIST Chemistry Webbook (https://webbook.nist.gov/chemistry/).

Acknowledgements

This work was supported by the Nation Natural Science Foundation of China (Grant No. 51925807, 52300051, 52192683, 52221004, 52170042) and China Postdoctoral Science Foundation (No. 2022M721811). Appreciation is also extended to the NSFC Innovative Research Groups Foundation (Grant No. 52221004). The femtosecond time-resolved transient absorption spectra were conducted at Yu-Xiang Weng’s laboratory in the Institute of Physics, CAS.

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Graphitic carbon nitride supported Fe single-atom nanozymes synergize with nitrate reductase for photobiocatalytic nitrate conversion

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Graphitic carbon nitride supported Fe single-atom nanozymes synergize with nitrate reductase for photobiocatalytic nitrate conversion

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Performance characterization of Fe/C₃N₄-CN/NarGH for nitrate conversion