Origin and Evolution of Biological Nitrogen Fixation

During Earth's Archean period, it has been suggested that high atmospheric concentrations of methane interfered with abiotic nitrogen fixation triggering a "fixed nitrogen crisis," limiting the proliferation of life. Fixed nitrogen limitation thereby provided strong selective pressure for the emergence of biological nitrogen fixation. In line with this rationale, recent isotopic evidence suggests that the primordial biosphere was severely limited in size before the evolution of biological nitrogen fixation, that the complex metalloenzyme responsible for biological nitrogen fixation, nitrogenase, pre-dates even oxygenic photosynthesis at 3.2 Gyr. The evolution of nitrogenase is intimately linked to photosynthesis and methanogenesis where homologs of nitrogenase (Nif) are critical enzymes is involved in the biosynthesis of chlorophyll (protochlorophyllide reductase or Bch/Chl) in photoautotrophs and cofactor F₄₃₀(Ni-sirohydrochlorin a,c-diamide reductive cyclase or Nfl) in methanogens. From previous phylogenetic work, it has been suggested that Nif and the enzyme Bch/Chl emerged from a common ancestor that resembled Nfl. We have put forth the hypothesis that the ancestral Nif bound metals and/or metal clusters in the substrate binding cavity of an Nfl-like ancestor. From a simplified binding cavity emerged via stepwise evolution complicated machinery with refined reactivity associated with the active site iron-molybdenum cofactor that we observe today in the contemporary nitrogenase. Analysis of ancestral sequence reconstructions provides a framework for assigning amino acid sequence determinants and functionalities that elucidates insights on the evolutionary relationships between nitrogen fixation, methanogenesis, and photosynthesis.