ATP-Triggered Fe(CN)<sub>2</sub>CO Synthon Transfer\nfrom the Maturase HypCD to the Active Site of Apo-[NiFe]-Hydrogenase

[NiFe]-hydrogenases catalyze the reversible activation\nof H<sub>2</sub> using a unique NiFe(CN)<sub>2</sub>CO metal site,\nwhich is\nassembled by a sophisticated multiprotein machinery. The [4Fe–4S]\ncluster-containing HypCD complex, which possesses an ATPase activity\nwith a hitherto unknown function, serves as the hub for the assembly\nof the Fe(CN)<sub>2</sub>CO subfragment. HypCD is also thought to\nbe responsible for the subsequent transfer of the iron fragment to\nthe apo-form of the catalytic hydrogenase subunit, but the underlying\nmechanism has remained unexplored. Here, we performed a thorough spectroscopic\ncharacterization of different HypCD preparations using infrared, Mössbauer,\nand NRVS spectroscopy, revealing molecular details of the coordination\nof the Fe(CN)<sub>2</sub>CO fragment. Moreover, biochemical assays\nin combination with spectroscopy, AlphaFold structure predictions,\nprotein–ligand docking calculations, and crosslinking MS deciphered\nunexpected mechanistic aspects of the ATP requirement of HypCD, which\nwe found to actually trigger the transfer of the Fe(CN)<sub>2</sub>CO fragment to the apo-hydrogenase.