Advancing nitrogenase research with asymmetry, hyperthermophiles, and semi-synthetic biomimetics – NITRO GENESE

Nitrogen-fixing technologies inspired by biology are required to produce the ammonia needed for today’s society in a green, carbon-neutral manner. Nitrogenase is the key to developing such approaches, being the only known enzyme capable of reducing kinetically inert dinitrogen (N2). It proceeds via a convoluted reaction requiring 16 equivalents of ATP to form two ammonia (NH3) equivalents. This N2-fixing machinery comprises the electron-providing Fe protein and a heterotetrameric MoFe protein harboring the P cluster and the FeMo-cofactor (FeMoco), the latter arguably being the most complex metallocofactor found in nature. Due to its organization and metallocofactors, fundamental questions remain to resolve many aspects of biological N2-fixation: (i) How do large motions and cooperativity in the MoFe protein affect its reactivity and Fe protein interaction? (ii) Where does N2 bind on the FeMoco? and (iii) how the protein matrix assists the multistep reaction taking place at the FeMoco? Elucidating this essential molecular process could lead to the creation of efficient bioinspired catalysts to produce green NH3 for fertilizers, chemical industries, and renewable energy storage. Here, NITRO GENESE seeks to address these central questions with a highly synergistic and unconventional toolkit comprising microbiology, biochemistry, biophysics, bioinformatics, spectroscopy, and inorganic chemistry, providing this consortium with an edge that would otherwise not be possible with individual research projects.

The question of cooperativity in nitrogenase will be addressed by preparing asymmetric, half-reactive MoFe proteins in a well-defined manner by tandem affinity chromatography using the model Azotobacter vinelandii. This will enable isolated MoFe protein halves to be studied with high confidence to understand how and why two halves of the MoFe protein engage in cooperativity and what the implications are for biological N2 fixation.

We will capitalize on the recent breakthrough of isolating a hyperthermostable MoFe protein that is catalytically stalled at room temperature, providing a critical springboard from which the N2-binding site and the NxHy intermediates can be resolved. Here, the MoFe protein is purified directly from an ancient archaeon living in deep-sea volcanoes. Additionally, characterizing the relatively simplified FeMoco synthetic machinery of the archaeon through genetic engineering will pave the way toward generating metallocofactor mimics and may later enable the production of active nitrogenase in eukaryotes, a highly attractive target for a fertilizer-free economy.

Finally, we will decipher the importance of the MoFe protein matrix to highly selective N2-transformation in an aqueous environment using semi-synthetic catalysts. First, a multitude of water-soluble FeMoco analogs will be synthesized chemically or biologically assisted. MoFe protein chassis devoid of the native FeMoco will host the mimic compounds to test their catalytic properties. In parallel, extracted FeMoco and mimics will be immobilized and activated for catalytic N2-electroreduction, a first proof-of-concept towards the use of bio-sourced electrocatalysts active sites, aiming at the electrification of ammonia synthesis.

Structural biology, spectroscopies, and computational modeling will act as the pillars of our approach, being essential to dissect the native and artificial metallocofactors concealed in the protein. Through such a combination of state-of-the-art techniques and skills, NITRO GENESE will elucidate the missing pieces of N2-biological fixation from the overall cooperative movements over the complete cycle to the atomic description of the catalytic chamber during N2 activation. The forthcoming groundbreaking results will enable the identification of the key elements required to design and implement upgraded bio- and electro-catalytic systems, laying the groundwork for carbon-neutral strategies for green ammonia production.