The Origins of ATP Dependence in Biological Nitrogen Fixation

Life depends on a conserved set of chemical energy currencies that are relics of early biochemistry. One of these is ATP, a molecule that, when paired with a divalent metal ion such as Mg ²⁺ , can be hydrolyzed to support numerous cellular and molecular processes. Despite its centrality to extant biochemistry, it is unclear whether ATP supported the function of ancient enzymes. We investigate the evolutionary necessity of ATP by experimentally reconstructing an ancestral variant of the key N ₂ -reducing enzyme nitrogenase. We show that the ancestor has a strict requirement for ATP and its hydrolysis is coupled to electron transfer for N ₂ reduction. Our results provide direct laboratory evidence of ATP usage by an ancient enzyme, and underscore how biomolecular constraints can entirely decouple cofactor selection from environmental availability.