Structural evolution of nitrogenase enzymes over geologic time

Abstract Life on Earth is about 4 billion years old—nearly as old as the planet itself. Over this immense timespan, living systems and their biomolecules have both adapted to and driven profound changes in the Earth’s environment. Among these, certain critical enzymes emerged early and have persisted through planetary extremes. Here, we implement an integrated approach to investigate the structural evolution of nitrogenase, an ancient and globally essential enzyme responsible for biological nitrogen fixation. Despite the ecological diversity of its host microbes, nitrogenase retains strict functional constraints, including extreme oxygen sensitivity, high energy demands, and substrate availability. We combined phylogenetics, ancestral sequence reconstruction, protein crystallography and deep-learning based structural prediction to resurrect nearly three billion years of nitrogenase structural history. This effort represents the first effort to predict the full set of extant and ancestral structures along the evolutionary tree of a single enzyme, yielding over 5000 structural models. Our framework lays a foundation for reconstructing key structural constraints that shape protein evolution and examining ancient enzymes within the broader context of phylogenetic relationships and environmental transitions across geological timescales.