Nitrogenase resurrection and the evolution of a singular enzymatic mechanism

Understanding how life’s earliest metabolic innovations emerged and coevolved with the planetary environment is a fundamental challenge for biology and key to unlocking novel bioengineering solutions. Protein resurrection studies can address this challenge through the development of tractable experimental systems to engineer historically consequential metabolisms. These systems must ultimately be tailored to reproduce key features of ancient proteins while permitting their functional evaluation within a complex, cellular and physiochemical context. Here, we present a phylogenetically informed engineering approach to resurrect and characterize ancient nitrogenases within an extant bacterial host. Ancestral nitrogenase genes were phylogenetically reconstructed and inserted into the genome of the diazotrophic bacterial model, Azotobacter vinelandii, enabling an integrated assessment of both in vivo functionality and purified nitrogenase biochemistry. We demonstrate that nitrogenase ancestors both enable diazotrophic growth of the host bacterium and catalyze dinitrogen reduction in vitro. Further, we find that the core dinitrogen reduction mechanism conserved by modern nitrogenases was established by the time the studied ancestors emerged. This mechanism is robust to the inferred, historical amino acid substitutions and variable incorporation of one or more ancestral protein subunits. Our results indicate that nitrogenase has been evolutionarily constrained by a single available mechanism for dinitrogen reduction, and has sustained this essential metabolic function over hundreds of millions of years.