Here we describe maturation of the [FeFe]-hydrogenase from its [4Fe-4S]-bound precursor state by using the synthetic complex [Fe2(μ-SH)2(CN)2(CO)4]2- together with HydF and components of the glycine cleavage system, but in the absence of the maturases HydE and HydG. This semisynthetic and fully-defined maturation provides new insights into the nature of H-cluster biosynthesis.