N2 fixation by the primary molybdenum (Mo)-nitrogenase requires traces of Mo. Yet, how microbes sustain this process under Mo scarcity is enigmatic. Project A10 investigates how the diazotrophic bacterium Rhodobacter capsulatus sustains nitrogen fixation when molybdenum (Mo) becomes limiting. The proposal challenges the paradigm that Mo-nitrogenase strictly depends on the Mo- containing active-site cofactor FeMoco and the NifEN maturase for activity. Preliminary data show that R. capsulatus can grow diazotrophically and reduce N₂ even when NifEN is deleted, and Mo import is restricted, suggesting that alternative or immature active-site metalloclusters may support residual nitrogenase function. The project has two main work packages. WP1 will comprehensively characterise distinct Mo-nitrogenase versions that share the same protein scaffold but differ in their active-site clusters. Their catalytic profiles will be compared using N₂, acetylene, and CO₂ reduction assays, while cryogenic electron microscopy, electron paramagnetic resonance spectroscopy, metal analysis, and homocitrate quantification will define structural and electronic differences between FeMoco and the alternative metalloclusters. WP2 will determine how extracellular and intracellular Mo concentrations regulate the switch between Mo- and iron-nitrogenase activity. This includes precisely controlling Mo levels in media, measuring nitrogenase expression and activity, developing intracellular molybdate biosensors, and linking nitrogen isotope fractionation patterns to specific nitrogenase versions. Overall, these studies will provide insights into survival strategies of diazotrophs in the race for Mo, redefine Mo dependence in bacterial nitrogen fixation, and reveal survival strategies under metal scarcity. Together with B01 “race for iron”, we will investigate and learn how microorganisms compete for metal ions under nutrient-limited conditions and in cross-kingdom communities. This understanding is key to design in MibiNet more complex microbial communities that race for metal ions, nitrogen, and carbon at the same time