Tracing the 'ninth sulfur' of the nitrogenase cofactor via a semi-synthetic approach

The M-cluster is the [(homocitrate)MoFe₇S₉C] active site of nitrogenase that is derived from an 8Fe core assembled viacoupling and rearrangement of two [Fe₄S₄] clusters concomitant with the insertion of an interstitial carbon and a 'ninth sulfur'. Combining synthetic [Fe₄S₄] clusters with an assembly protein template, here we show that sulfite can give rise to the ninth sulfur that is incorporated in the catalytically important belt region of the cofactor after the radical S-adenosyl-L-methionine-dependent carbide insertion and the concurrent 8Fe-core rearrangement have already taken place. Based on the differential reactivity of the formed cluster species, we also propose a new [Fe₈S₈C] cluster intermediate, the L*-cluster, which is similar to the [Fe₈S₉C] L-cluster, but lacks the ninth sulfur from sulfite. This work provides a semi-synthetic tool for protein reconstitution that could be widely applicable for the functional analysis of other FeS systems.