Abstract:

Nitrogenase catalyzes biological nitrogen fixation: the reduction of inert dinitrogen to bioavailable ammonia. This process is vital to the global nitrogen cycle, and features exciting chemistry with promise of new biological and synthetic paradigms. This presentation covers my work validating the activity of nitrogenase proteins expressed in E. coli, and using this platform to study the biological synthesis of the catalytic FeMo cofactor precursor and its activity in maturation proteins NifB and NifEN. I will present chemical evidence from both in-vivo and in-vitro assays with purified proteins and structural evidence from EPR and EXAFS spectroscopy. Using mutational studies, we have found key ligands coordinate 3 Fe₄S₄ clusters in the radical SAM enzyme NifB. These mutants have varying SAM cleaving ability and nitrogenase maturation activity, providing insights into nitrogenase cofactor maturation. The resulting unmatured cofactor is passed to either the NifEN scaffold protein, or VnfEN vanadium dependent analogue if molybdenum is absent. Our work shows that there is low activity of substrate reduction directly from the EN scaffolds with the precursor cofactor, with NifEN having both higher activity and a more structurally dynamic Fe₄S₄ cluster. These studies will inform future efforts of bioengineering nitrogenase for agricultural and industrial applications and expand understanding of chalcogenic complexes.