Biochemical characterization of the iron nitrogenase

Nitrogen is an essential building block for life. Its biologically relevant reduced form is mainly derived from dinitrogen (N2) through the industrial Haber-Bosch process and the natural nitrogen cycle. In the latter, a class of highly sophisticated metalloenzymes known as nitrogenases catalyze the reduction of N2 to ammonia (NH3) under biological conditions. Three nitrogenase isoforms are known to date, which are classified according to the metal content of their active site cofactor: the molybdenum (Mo), vanadium (V) and iron (Fe) nitrogenase. Even though these enzymes are best known for their ability to break the stable triple bond of the N2 molecule, previous studies have revealed an increasing amount of promiscuous nitrogenase activities. Remarkably, these include the reduction of the greenhouse gas carbon dioxide (CO2), a reaction that offers novel perspectives for the recycling of carbon waste. Among the three nitrogenase isozymes, the Fe nitrogenase displays the highest wild-type efficiency for the reduction of CO2, but the molecular mechanisms facilitating this activity remain elusive. The results presented in this work provide a fundamental biochemical characterization of the Fe nitrogenase and aim to unravel the molecular properties that facilitate the reduction of N2 and CO2. Thereby, this work establishes a framework to leverage the remarkable properties of the Fe nitrogenase for biotechnological applications. Specifically, this thesis provides insights into the Fe nitrogenase reactivity (Chapter III), molecular structure (Chapter IV) and maturation pathway (Chapter V). Initially, Chapter II introduces the three nitrogenase isozymes, elucidates on the current knowledge of the nitrogenase molecular mechanism and provides context for their promiscuous activities. Chapter III presents the results of a comparative study on the Fe and Mo nitrogenases of Rhodobacter capsulatus, where we analyzed their competing reduction of N2 and CO2. By monitoring the in vitro CO2 reduction activity in the presence of N2, we find the Fe nitrogenase to be highly promiscuous for the reduction of CO2, which results in the in vitro formation of formate, carbon monoxide and methane. We further demonstrate that these effects translate in vivo, where we observe an extracellular accumulation of the same three products by Fe nitrogenase expressing strains upon exposure to CO2. Chapter IV elucidates on the molecular foundation of these reactivities through a 2.35 Å cryogenic electron microscopy structure of the Fe nitrogenase complex. The structure reveals the nature of the Fe nitrogenase active site cofactor and highlights a Fe nitrogenase specific complex architecture. Chapter V introduces AnfG - a so far uncharacterized subunit of the Fe nitrogenase complex - as an essential factor in the Fe nitrogenase maturation pathway and provides molecular insights into the Fe nitrogenase apoenzyme. Finally, Chapter VI puts the presented results into a larger context and discusses future research directions.