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

Molybdenum nitrogenase catalyses the ambient reduction of N-2 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 N-2 via displacement of the belt-sulfur(s) of the M-cluster upon turnover. However, direct proof of N-2 binding and belt-sulfur mobilization during catalysis remains elusive. Here we show that N-2 is captured on the M-cluster via electron and sulfur depletion, and that the N-2-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.

Automated summary

This study provides direct evidence that N2 is captured on the M-cluster via electron and sulfur depletion, and the N2-captured state is catalytically active in generating NH3. The study also reveals the conditions for product release and the dynamic changes of belt-sulfurs during catalysis. These findings highlight the crucial role of the mobilization of cofactor belt-sulfurs in the nitrogenase reaction.