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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.

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Fig. 1: Assembly of the M-cluster.
Fig. 2: DT-dependent cluster maturation.
Fig. 3: DT-free cluster maturation.
Fig. 4: Refined model of L-cluster assembly on NifB.


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This work was supported by NIH-NIGMS grant GM67626 (to M.W.R. and Y.H.), DOE-BES grant DE-DC0014470 (to M.W.R. and Y.H.), a Takeda Science Foundation grant (to Y.O.) and Grant-in-Aids for Scientific Research (nos 23000007 and 16H04116) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to K.Tat. and Y.O.).

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K.Tan., C.C.L., N.S.S., Y.H. and M.W.R. designed and analysed experiments. K.Tan. performed experiments. K.Tat. and Y.O. provided materials. Y.H. and M.W.R. wrote the manuscript with input from all the authors.

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Correspondence to Yilin Hu or Markus W. Ribbe.

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Supplementary Methods, References, and Figures 1–6

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Tanifuji, K., Lee, C.C., Sickerman, N.S. et al. Tracing the ‘ninth sulfur’ of the nitrogenase cofactor via a semi-synthetic approach. Nature Chem 10, 568–572 (2018).

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