Nitrogenases are a group of complex metalloenzymes that convert molecular nitrogen (N₂) to its bioavailable form ammonia (NH₃). Thus, they play a critical role in biological nitrogen fixation. In general, there are three nitrogenase isoforms in diazotrophic microorganisms. The most common/prevalent nitrogenase is the molybdenum (Mo) nitrogenase and the less abundant vanadium (V) and iron (Fe) nitrogenases, named after the metal composition of the active site cofactor. All three nitrogenase isoforms are known as bona fide nitrogenases. However, there are several nitrogen fixation-like (Nfl)-enzymes that share sequence similarities and conserved regions but lack the ability to fix dinitrogen. The distinct activities of all those enzymes are attributed to differences in their catalytic metal center. The activities of those Nfl-like enzymes range from bacteriochlorophyll synthesis (dark-operative protochlorophyllide oxidoreductase and chlorophyllide a oxidoreductase, reducing carbon-carbon double bonds of the tetrapyrrole rings) (Moser & Layer, Methods Mol Biol, 2019), F430 biosynthesis (Ni2+-sirohydrochlorin a,c-diamide reductase) (Vazquez Ramos et al., FEBS J, 2024 / Moore et al., Nature, 2017) and methylated sulfur compounds (North et al., Science, 2020 / Lago-Maciel et al., bioRxiv, 2024).

However, in contrast to the type Nfl-enzymes described above, another type IV homolog from Endomicrobium proavitum has recently been shown to reduce N₂ to NH₃ in vivo (Zheng et al., Environ. Microbiol., 2016). Following up on this surprising finding, we set out to comprehensively characterize this enzyme. I am currently investigating its atomic structure using cryo-electron microscopy, determining the metal content of its cofactors by inductively coupled plasma optical emission spectroscopy, and exploring its substrate spectrum in vitro. Our exciting findings will provide new insights into the inner workings of Nfl-enzymes and nitrogenases, potentially revealing the fundamental principles of N₂-reduction and the different substrate specificity of those enzymes.