Emergence of an Orphan Nitrogenase Protein Following Atmospheric Oxygenation

Mol Biol Evol. 2024 Apr 2;41(4):msae067. doi: 10.1093/molbev/msae067.


Molecular innovations within key metabolisms can have profound impacts on element cycling and ecological distribution. Yet, much of the molecular foundations of early evolved enzymes and metabolisms are unknown. Here, we bring one such mystery to relief by probing the birth and evolution of the G-subunit protein, an integral component of certain members of the nitrogenase family, the only enzymes capable of biological nitrogen fixation. The G-subunit is a Paleoproterozoic-age orphan protein that appears more than 1 billion years after the origin of nitrogenases. We show that the G-subunit arose with novel nitrogenase metal dependence and the ecological expansion of nitrogen-fixing microbes following the transition in environmental metal availabilities and atmospheric oxygenation that began ∼2.5 billion years ago. We identify molecular features that suggest early G-subunit proteins mediated cofactor or protein interactions required for novel metal dependency, priming ancient nitrogenases and their hosts to exploit these newly diversified geochemical environments. We further examined the degree of functional specialization in G-subunit evolution with extant and ancestral homologs using laboratory reconstruction experiments. Our results indicate that permanent recruitment of the orphan protein depended on the prior establishment of conserved molecular features and showcase how contingent evolutionary novelties might shape ecologically important microbial innovations.

Keywords: ancestral sequence reconstruction; early life and evolution; nitrogen fixation; nitrogenase; orphan genes; planetary biology.

MeSH terms

  • Nitrogen / metabolism
  • Nitrogen Fixation* / genetics
  • Nitrogenase* / chemistry
  • Nitrogenase* / genetics
  • Nitrogenase* / metabolism


  • Nitrogenase
  • Nitrogen