(Invited) ATP-Independent Electroenzymatic Ammonia Production Using an Organic Redox Polymer-Immobilized Enzymatic System

Consuming approximately 1% of the total fossil fuel of the world, the ammonia (NH 3 ), a usable form to living organisms is produced by dinitrogen (N 2 ) fixation. Currently, approximately 66% of NH 3 is produced by the Haber–Bosch process using an iron catalyst with N 2 and H 2 at high temperatures and high pressures (400 °C and 20 MPa), which are estimated to be responsible for approximately 3% of CO 2 emissions. 1,2 As an alternative strategy, bioelectrosynthetic technique is studied for eco-friendly ammonia production using the nitrogenase, the only enzyme able to reduce N 2 to NH 3 at room temperature, neutral pH and ambient pressure. For the practical use of nitrogenase bioelectrocatalysis technology, it is indispensable to minimize the large input of chemical energy for the reduction of MoFe protein component of nitrogenase by hydrolysis of two ATP for each electron transferred (16 ATP per N 2 ). 3-5 Therefore, we have concentrated on approaches to bypass the reducing- and ATP-hydrolyzing properties of the dinitrogenase reductase by immobilizing the catalytic protein of nitrogenase, MoFe protein, on the electrode surface. One way to directly reduce the MoFe protein is a use of a redox polymer having thermodynamically strong reducing capability. Redox polymer immobilized bioelectrocatalysis facilitates an efficient electron transfer through self-exchange based conduction due to a high effective concentration of catalysts at the electrode surface and more efficient mediation. However, none of the previously reported redox polymers were capable of immobilized bioelectrocatalysis using nitrogenase. 6-9 Thus, we report the MoFe nitrogenase immobilized at an electrode surface with a neutral red redox polymer which is used to reduce the MoFe protein (as mediator able to transfer the electrons to the MoFe independent of the Fe protein and of ATP hydrolysis) and support the mediated bioelectrocatalysis of N 3 - , NO 2 - and N 2 to NH 3 catalyzed by the MoFe protein. Representative Bulk bioelectrosynthetic experiments produced 209 ± 30 nmol NH 3 nmol MoFe -1 h -1 from N 2 reduction. 15 N 2 labeling experiments and NMR analysis were performed to confirm biosynthetic N 2 reduction to NH 3 . Reference K. Burgess and D. J. Lowe, Chem. Rev. , 1996, 96, 2983–3012. Christiansen, D. R. Dean and L. C. Seefeldt, Annu. Rev. Plant Physiol. Plant Mol. Biol. , 2001, 52, 269–295. E. Smith, Science , 2002, 297, 1654–1655. -Y. Yang, K. Danyal and L. C. Seefeldt, in Nitrogen Fixation, Methods in Molecular Biology 766 , ed. M. W. Ribbe, Springer, New York, 2011, ch. 2. Milton, R. D.; Abdellaoui, S.; Khadka, N.; Dean, D. R.; Leech, D.; Seefeldt, L. C.; Minteer, S. D. Energy Environ. Sci ., 2016, 9, 2550– 2554 Hickey, D. P.,Cai, R., Yang, Z. Y., Grunau, K., Einsle, O., Seefeldt, L. C., Minteer, S. D., Am. Chem. Soc ., 2019, 141, 17150– 17157 Ackermann, D. A. Guschin, K. Eckhard, S. Shleev, W. Schuhmann, Electrochem Comm ., 2010, 12, 640-643. Barrière, Y. Ferry, D. Rochefort, D. Leech, Electrochem Comm. , 2004, 6, 237-241 Yuan, S. D. Minteer, Curr. Opin. Electrochem., 2019, 15, 1-6