The M-cluster is the active site of nitrogenase that is derived from an 8Fe-core (designated L-cluster) assembled via coupling and rearrangement of two [Fe4S4] clusters concomitant with the insertion of an interstitial carbon and a ‘9th sulfur’. Combining synthetic [Fe4S4] clusters with an assembly protein template, we showed that sulfite gives rise to the ‘9th 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. Based on these findings, we proposed an [Fe8S8C] cofactor precursor (designated the L*-cluster) that is geometrically indistinguishable from the [Fe8S9C] L-cluster but lacks the ‘9th sulfur’ in the belt region of the L-cluster. Detailed x-ray absorption spectroscopy and electron spin echo envelope modulation (ESEEM) analyses confirms the existence of the L*-cluster and the structural homology of this cluster to the L-cluster except for the missing belt sulfur. This work provides a semi-synthetic tool for strategically labeling the cofactor—including its ‘9th S’ in the belt region—for mechanistic investigations of nitrogenase while suggesting an interesting link between nitrogen fixation and sulfite detoxification in diazotrophic organisms.

Nitrogenase M-cluster assembly

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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