Thomas B. Rauchfuss

The paper aims to elucidate the final stages in the biosynthesis of the [2Fe] H active site of the [FeFe]‐hydrogenases. The recently hypothesized intermediate [Fe 2 (SCH 2 NH 2 ) 2 (CN) 2 (CO) 4 ] 2− ([ 1 ] 2− ) was prepared by a multistep route from [Fe 2 (S 2 )(CN)(CO) 5 ] − . The following synthetic intermediates were characterized in order: [Fe 2 (SCH 2 NHFmoc) 2 (CNBEt 3 )(CO) 5 ] − , [Fe 2 (SCH 2 NHFmoc) 2 (CN)−(CO) 5 ] − , and [Fe 2 (SCH 2 NHFmoc) 2 (CN) 2 (CO) 4 ] 2− , where Fmoc is fluorenylmethoxycarbonyl). Derivatives of these anions include [K(18‐crown‐6)] + , PPh 4 + and PPN + salts as well as the 13 CD 2 ‐isotopologues. These Fe 2 species exist as a mixture of two isomers attributed to diequatorial (ee) and axial‐equatorial (ae) stereochemistry at sulfur. In vitro experiments demonstrate that [ 1 ] 2− maturates HydA1 in the presence of HydF and a cocktail of reagents. HydA1 can also be maturated using a highly simplified cocktail, omitting HydF and other proteins. This result is consistent with HydA1 participating in the maturation process and refines the roles of HydF.