The nitrogenase G-subunit is an ancient orphan protein that drove the ecological expansion of nitrogen fixation

ABSTRACT Nitrogenase metalloenzymes have catalyzed biological nitrogen fixation for billions of years and revolutionized planet Earth by supplying essential nitrogen to the biosphere. How these enzymes were built and distributed by microbial and evolutionary processes in a shifting geochemical landscape remains an open question. Here, we probe the birth and evolution of the G-subunit protein, an integral, Precambrian-age structural component of certain nitrogenase isozymes that makes its appearance midway through nitrogenase evolutionary history. We establish that the G-subunit is an orphan protein, with no homologs detected across wider protein diversity. We find that G-subunit emergence accompanied both the diversification of nitrogenase metal usage and an ecological expansion of nitrogen-fixing microbes during the transition in enviromental metal availabilities triggered by Earth surface oxygenation ∼2.5 billion years ago. Further, analyses of ancestral nitrogenase structures implicate a role for the G-subunit in novel metal incorporation, which would have primed nitrogenases and their hosts to exploit these newly diversified geochemical environments. However, permanent recruitment of the G-subunit into the nitrogenase complex was likely only enabled by tuning preexisting, protein interaction features that were selected prior to Earth oxygenation. Our results showcase how contingent evolutionary novelties shape microbial ecological responses and their global consequences.