Synthesis of diiron subcluster [Fe2[(μ-SCH2)2NH](CN)2(CO)4]2- for artificial maturation of [FeFe]-hydrogenases.

The [FeFe]-hydrogenases are widely distributed enzymes that efficiently make and use hydrogen (H2). They have attracted intense interest as potential biocatalysts as well as models for biomimetic catalysis. Substrate turnover occurs at an Fe-based active site featuring an azadithiolate cofactor. The biosynthesis of the active site is remarkably elaborate. The production of these enzymes is greatly simplified by replacing the biosynthesis of this active site with reconstitution of the enzyme with chemically synthesized [Fe2[(μ-SCH2)2NH](CN)2(CO)4]2- ([1]2-), the subject of this protocol. Two approaches to [1]2- are described: a 'condensation route' and an 'Fmoc route'. Each of the two routes requires ~20 h of hands-on time. The condensation route entails the reaction of Fe2(μ-SH)2(CO)6 and N4(CH2)6 followed by cyanation to give [1]2-. The most demanding step in this route is the preparation of Fe2(μ-S2)(CO)6. The second route focuses on fluorenylmethoxycarbonyl (Fmoc)-protected derivatives of azadithiolate cofactor. This route proceeds via Fe2[(μ-SCH2)2NFmoc](CO)6. Both the condensation and Fmoc routes allow isotopic labeling of the active site, but 57Fe labeling is especially simple using the Fmoc route.