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[NiFe] hydrogenases catalyze the reversible oxidation of molecular hydrogen into two protons and two electrons. A key organometallic chemistry feature of the NiFe active site is that the iron atom is co-coordinated by two cyanides (CN^–) and one carbon monoxide (CO) ligand. Biosynthesis of the NiFe(CN)₂(CO) cofactor requires the activity of at least six maturation proteins, designated HypA–F. An additional maturase, HypX, is required for CO ligand synthesis under aerobic conditions, and preliminary in vivo data indicated that HypX releases CO using N¹⁰-formyltetrahydrofolate (N¹⁰-formyl-THF) as the substrate. HypX has a bipartite structure composed of an N-terminal module similar to N¹⁰-formyl-THF transferases and a C-terminal module homologous to enoyl-CoA hydratases/isomerases. This composition suggested that CO production takes place in two consecutive reactions. Here, we present in vitro …