Molecular basis of N2 fixation in a hyperthermophilic archaeon.

Exploring the natural diversity of phylogenetically distant nitrogenases is crucial for gaining new insights into the mechanism of atmospheric N2 fixation and unlocking biotechnological developments in sustainable ammonia production. Here, we investigated the N2-fixing system of Methanocaldococcus infernus, a deep-sea hyperthermophilic archaeon growing diazotrophically at 92 °C. This natively isolated nitrogenase has a melting temperature close to the water boiling point, with an extrapolated specific activity superior to mesophilic homologues. The crystal structures obtained at near-atomic resolution present the most simplified known nitrogenase, harbouring strategic hot spots for thermostability. It combines the structural traits of all three known nitrogenase isoforms, reinforcing the proposal that ancestral nitrogenases were more similar to the archaeal enzyme than to the bacterial homologues. In contrast to structural homologues, the electron-transferring metallocofactor "P-cluster" is trapped in a rare state awaiting electron delivery, providing a detailed picture of the physiological state. The active site, harbouring the FeMo-cofactor catalyst, exhibits a mixture of the resting and "turnover" states, previously described solely in the bacterial vanadium and iron-only nitrogenases. Therefore, these results unify a mechanistic principle of all nitrogenases and highlight the advantages of the hyperthermostable nature of the archaeal enzyme, opening new avenues for further understanding of how nature splits the N2 triple bond.