Abstract
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.