Final Stages in the Biosynthesis of the [FeFe]-Hydrogenase Active Site

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 [Fe₂(SCH₂NH₂)₂(CN)₂(CO)₄]^2- ([1]^2-) was prepared by a multistep route from [Fe₂(S₂)(CN)(CO)₅]^-. The following synthetic intermediates were characterized in order: [Fe₂(SCH₂NHFmoc)₂(CNBEt₃)(CO)₅]^-, [Fe₂(SCH₂NHFmoc)₂(CN)-(CO)₅]^-, and [Fe₂(SCH₂NHFmoc)₂(CN)₂(CO)₄]^2-, where Fmoc is fluorenylmethoxycarbonyl). Derivatives of these anions include [K(18-crown-6)]⁺, PPh₄ ⁺ and PPN⁺ salts as well as the ¹³CD₂-isotopologues. These Fe₂ 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.