Investigating the Molybdenum Nitrogenase Mechanistic Cycle Using Spectroelectrochemistry.

Molybdenum nitrogenase plays a crucial role in the biological nitrogen cycle by catalyzing the reduction of dinitrogen (N) to ammonia (NH) under ambient conditions. However, the underlying mechanisms of nitrogenase catalysis, including electron and proton transfer dynamics, remain only partially understood. In this study, we covalently attached molybdenum nitrogenase (MoFe) to gold electrodes and utilized surface-enhanced infrared absorption spectroscopy (SEIRA) coupled with electrochemistry techniques to investigate its catalytic mechanism. Our biohybrid system enabled electron transfer via a mild mediator, likely mimicking the natural electron flow through the P-cluster to FeMoco, the enzyme's active site. For the first time, we experimentally observed both terminal and bridging S-H stretching frequencies, resulting from the protonation of bridging sulfides in FeMoco during turnover conditions providing direct evidence of their role in catalysis. These experimental observations are further supported by QM/MM calculations. Additionally, we investigated CO inhibition, demonstrating both CO binding and unbinding dynamics under electrochemical conditions. These insights not only advance our understanding of the mechanistic cycle of molybdenum nitrogenase but also establish a foundation for studying alternative nitrogenases, including vanadium and iron nitrogenases.