Photocatalytic Removal of the Greenhouse Gas Nitrous Oxide by Liposomal Microreactors

Abstract

Nitrous oxide (N₂ O) is a potent greenhouse and ozone-reactive gas for which emissions are growing rapidly due to increasingly intensive agriculture. Synthetic catalysts for N₂ O decomposition typically contain precious metals and/or operate at elevated temperatures driving a desire for more sustainable alternatives. Here we demonstrate self-assembly of liposomal microreactors enabling catalytic reduction of N₂ O to the climate neutral product N₂ . Photoexcitation of graphitic N-doped carbon dots delivers electrons to encapsulated N₂ O Reductase enzymes via a lipid-soluble biomolecular wire provided by the MtrCAB protein complex. Within the microreactor, electron transfer from MtrCAB to N₂ O Reductase is facilitated by the general redox mediator methyl viologen. The liposomal microreactors use only earth-abundant elements to catalyze N₂ O removal in ambient, aqueous conditions.

Keywords: Carbon Dot; Enzyme Catalysis; Liposomes; Nitrous Oxide; Photochemistry.

Figure 1

MtrCAB and its Role in Liposomal Microreactors for N₂O Removal. A) Model for the MtrCAB complex from S. oneidensis based on the crystal structure ^[7c] of S. baltica MtrCAB. Hemes (orange) with iron (black) are shown as spheres within the MtrA and MtrC proteins (yellow). The MtrA heme chain is insulated from the membrane by embedding within a beta‐barrel formed by MtrB (gray) for which the front surface is not shown. MtrA and MtrB are assembled as a naturally insulated biomolecular nanowire with both structural and functional attributes analogous to those of an electrical power cable. B) Schematic of a liposome microreactor with N₂O Reductase encapsulated within a lipid bilayer membrane spanned by MtrCAB. Diagram not to scale and is purely to aid discussion, the orientation of MtrCAB is not experimentally defined. Panel B created with BioRender.com.

Figure 2

Characterization of Proteoliposomes. A) Electronic absorbance of N₂O Reductase containing proteoliposomes with (red continuous line) and without (black continuous line)MtrCAB. Proteoliposomes (≈3 nM) in 50 mM Tris‐HCl, 10 mM KCl, pH 8.5. Circles show the estimated contribution to each spectrum from proteoliposome scattering, see Supporting Information for details. B) Coomassie stained SDS‐PAGE gel image for N₂O Reductase containing proteoliposomes without (center lane) and with (right lane) MtrCAB. Molecular weight markers of the indicated mass (left lane).

Figure 3

Dithionite‐Driven Proteoliposome N₂O Reductase Activity. Electronic absorbance spectra for suspensions of N₂O Reductase containing proteoliposomes with (A) and without (B) MtrCAB measured for 12 min after addition of 750 μM N₂O at t=0 min. Arrows indicate the direction of spectral change for the features corresponding to sodium dithionite (315 nm) and MV⋅⁺ (395 nm). For (A) spectra at t = 0 (thick line), 1, 3, 5, 7, 10 and 11 (thin lines) min. For (B) spectra at t = 0 (thick line), 1, 3, 7, 8, and 11 (thin lines) min. Proteoliposomes (≈3 nM) in anaerobic 100 μM dithionite, 10 μM MV, 50 mM Tris‐HCl, 10 mM KCl, pH 8.5. Spectra are presented after subtraction of scattering due to proteoliposomes; see Supporting Information for details. Time course for oxidation of dithionite (C) and MV⋅⁺ (D) by N₂O Reductase containing proteoliposomes with (red) and without (black) MtrCAB. In the presence of MtrCAB, after ≈9 min the dithionite is depleted which results in rapid oxidation of MV⋅⁺. Data are the average of n=3 datasets with error bars as standard deviation.

Figure 4

The Role of Methyl Viologen (MV) in MtrCAB Supported Proteoliposome N₂O Reduction. Dithionite or irradiated carbon dots reduce MV²⁺ to bilayer permeable MV⋅⁺. Inside the proteoliposome MV⋅⁺ driven N₂O reduction is catalyzed by N₂O Reductase (blue) regenerating MV²⁺. A) In the absence of MtrCAB the MV²⁺ is trapped inside the liposome. B) In the presence of MtrCAB (orange) electrons from external (photo)reductants enter the liposome via the protein biowire and re‐reduce encapsulated MV²⁺. This process drives further N₂O reduction. With N₂O in excess of dithionite, when the latter becomes fully oxidized the MV is converted to MV²⁺ trapped inside the liposomes. Diagram not to scale and is purely to aid discussion, the orientation of MtrCAB is not experimentally defined. Created with BioRender.com.

Figure 5

Gas Chromatographic Analysis of N₂O Reduction. The difference in headspace N₂O concentration is presented for A) suspensions of N₂O Reductase containing proteoliposomes with and without MtrCAB, and B) solutions with and without free N₂O Reductase. A) Anaerobic vials with N₂O (1.5 μmol total) in 1 mL headspace and 2 mL of 100 μg mL⁻¹ graphitic N‐doped carbon dots, 10 μM MV, 3 nM proteoliposomes, 25 mM EDTA, 50 mM Tris‐HCl, 10 mM KCl, pH 8.5. Proteoliposomes (8 nM) were introduced at t=0 hr. Irradiation with visible light (2.5 kW m⁻²) for 4 hr was followed by 4 hr in dark. Circles show the average of n=3 datasets with error bars as standard deviation. B) Anaerobic vials with N₂O (1.5 μmol total) in 1 mL headspace and 2 mL of 1600 μM MV, 800 μM dithionite, 50 mM Tris:HCl, 10 mM KCl, pH 8.5. N₂O Reductase (150 nM) was added to half the vials at t=0 hr. For these conditions complete removal of N₂O was expected in 5 min when N₂O Reductase was present, see Supporting Information and Figure S6 for further details.PLEASE REPLACE THE EXISTING TOC IMAGE with the one below.