Reconstructing the evolutionary history of nitrogenases: Evidence for ancestral molybdenum-cofactor utilization

Amanda K. Garcia, Hanon McShea, Bryan Kolaczkowski, Betül Kaçar

Research output: Contribution to journalArticlepeer-review

36 Scopus citations


The nitrogenase metalloenzyme family, essential for supplying fixed nitrogen to the biosphere, is one of life's key biogeochemical innovations. The three forms of nitrogenase differ in their metal dependence, each binding either a FeMo-, FeV-, or FeFe-cofactor where the reduction of dinitrogen takes place. The history of nitrogenase metal dependence has been of particular interest due to the possible implication that ancient marine metal availabilities have significantly constrained nitrogenase evolution over geologic time. Here, we reconstructed the evolutionary history of nitrogenases, and combined phylogenetic reconstruction, ancestral sequence inference, and structural homology modeling to evaluate the potential metal dependence of ancient nitrogenases. We find that active-site sequence features can reliably distinguish extant Mo-nitrogenases from V- and Fe-nitrogenases and that inferred ancestral sequences at the deepest nodes of the phylogeny suggest these ancient proteins most resemble modern Mo-nitrogenases. Taxa representing early-branching nitrogenase lineages lack one or more biosynthetic nifE and nifN genes that both contribute to the assembly of the FeMo-cofactor in studied organisms, suggesting that early Mo-nitrogenases may have utilized an alternate and/or simplified pathway for cofactor biosynthesis. Our results underscore the profound impacts that protein-level innovations likely had on shaping global biogeochemical cycles throughout the Precambrian, in contrast to organism-level innovations that characterize the Phanerozoic Eon.

Original languageEnglish (US)
Pages (from-to)394-411
Number of pages18
Issue number3
StatePublished - May 1 2020


  • ancestral sequence reconstruction
  • metal cofactor
  • metalloenzyme
  • nitrogen fixation
  • nitrogenase

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Environmental Science(all)
  • Earth and Planetary Sciences(all)


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