Abstract: | Nitrogenases catalyze the reduction of N2 to NH4+ at its cofactor site. Designated the M‐cluster, this MoFe7S9C(R‐homocitrate)] cofactor is synthesized via the transformation of a Fe4S4] cluster pair into an Fe8S9C] precursor (designated the L‐cluster) prior to insertion of Mo and homocitrate. We report the characterization of an eight‐iron cofactor precursor (designated the L*‐cluster), which is proposed to have the composition Fe8S8C] and lack the “9th sulfur” in the belt region of the L‐cluster. Our X‐ray absorption and electron spin echo envelope modulation (ESEEM) analyses strongly suggest that the L*‐cluster represents a structural homologue to the l ‐cluster except for the missing belt sulfur. The absence of a belt sulfur from the L*‐cluster may prove beneficial for labeling the catalytically important belt region, which could in turn facilitate investigations into the reaction mechanism of nitrogenases. |