Nitrogenase M-Cluster Assembly: Tracing the ‘9th Sulfur’ of the Nitrogenase Cofactor via a Semi-Synthetic Approach

The M-cluster is the active site of nitrogenase that contains an 8Fe-core assembled via coupling and rearrangement of two [Fe₄S₄] clusters concomitant with the insertion of an interstitial carbon and a ‘9^th sulfur’. Combining synthetic [Fe₄S₄] clusters with an assembly protein template, we show that sulfite gives rise to the ‘9^th sulfur’ that is incorporated in the catalytically important belt region of the cofactor after the radical SAM-dependent carbide insertion and the concurrent 8Fe-core rearrangement have already taken place. This work provides a semi-synthetic tool for strategically labeling the cofactor—including its ‘9^th S’ in the belt region—for mechanistic investigations of nitrogenase while suggesting an interesting link between nitrogen fixation and sulfite detoxification in diazotrophic organisms.