Evidence of substrate binding and product release via belt-sulfur mobilization of the nitrogenase cofactor

Molybdenum nitrogenase catalyses the ambient reduction of N2 to NH3 at the M-cluster, a complex cofactor that comprises two metal-sulfur partial cubanes ligated by an interstitial carbide and three belt-sulfurs. A recent crystallographic study suggests binding of N2 via displacement of the belt-sulfur(s) of the M-cluster upon turnover. However, direct proof of N2 binding and belt-sulfur mobilization during catalysis remains elusive. Here we show that N2 is captured on the M-cluster via electron and sulfur depletion, and that the N2-captured state is catalytically competent in generating NH3. Moreover, we demonstrate that product release occurs only when sulfite is supplied along with a reductant, that sulfite is inserted as sulfide into the belt-sulfur-displaced positions and that there is a dynamic in-and-out of belt-sulfurs during catalysis. Together, these results establish the mobilization of cofactor belt-sulfurs as a crucial, yet overlooked, mechanistic element of the nitrogenase reaction. A recently proposed structure of an N2-bound Mo-nitrogenase has sparked considerable attention, although the direct evidence for N2 binding and sulfur mobilization during turnover has remained elusive. Now, additional spectroscopic and kinetic measurements further support this state and provide evidence that belt-sulfur displacement is an essential aspect of the nitrogenase mechanism.