Maturation of [FeFe]-hydrogenase (HydA) involves synthesis of a CO, CN À , and dithiomethylamine (DTMA)coordinated 2Fe subcluster that is inserted into HydA to make the active hydrogenase. This process requires three maturation enzymes: the radical S-adenosyl-L-methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. In vitro maturation with purified maturation enzymes has been possible only when clarified cell lysate was added, with the lysate presumably providing essential components for DTMA synthesis and delivery. Here we report maturation of [FeFe]-hydrogenase using a fully defined system that includes components of the glycine cleavage system (GCS), but no cell lysate. Our results reveal for the first time an essential role for the aminomethyl-lipoyl-H-protein of the GCS in hydrogenase maturation and the synthesis of the DTMA ligand of the H-cluster. In addition, we show that ammonia is the source of the bridgehead nitrogen of DTMA.

The [FeFe]-hydrogenase H-cluster is a complex organometallic cofactor whose assembly and installation requires three dedicated accessory proteins referred to as HydE, HydF, and HydG. The roles of these maturases and the precise mechanisms by which they synthesize and insert the H-cluster are not fully understood. This Minireview will focus on new insights into the [FeFe]-hydrogenase maturation process that have been provided by in vitro approaches in which the biosynthetic pathway has been partially or fully reconstructed using semisynthetic and enzyme-based approaches. Specifically, the application of these in vitro, semisynthetic, and fully defined approaches has shed light on the roles of individual maturation enzymes, the nature of H-cluster assembly intermediates, the molecular precursors of H-cluster ligands, and the sequence of steps involved in [FeFe]-hydrogenase maturation.

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Die Bildung einer aktiven [FeFe]‐Hydrogenase , des effizientesten Enzyms für die H2‐Produktion, erfordert die Synthese eines komplexen Metallzentrums, des H‐Clusters, der für die reversible Reduktion von Protonen zu H2 verantwortlich ist. In ihrer Zuschrift (e202203413) zeigen Joan B. Broderick und Mitarbeiter, dass der letzte Schritt dieser Synthese, der aus dem Aufbau des Azadithiolat‐Brückenmoleküls besteht, das Aminomethyl‐Lipoyl‐H‐Protein aus dem Glycinspaltsystem einbezieht.

Maturation of [FeFe]-hydrogenase (HydA) involves synthesis of a CO, CN À , and dithiomethylamine (DTMA)coordinated 2Fe subcluster that is inserted into HydA to make the active hydrogenase. This process requires three maturation enzymes: the radical S-adenosyl-L-methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. In vitro maturation with purified maturation enzymes has been possible only when clarified cell lysate was added, with the lysate presumably providing essential components for DTMA synthesis and delivery. Here we report maturation of [FeFe]-hydrogenase using a fully defined system that includes components of the glycine cleavage system (GCS), but no cell lysate. Our results reveal for the first time an essential role for the aminomethyl-lipoyl-H-protein of the GCS in hydrogenase maturation and the synthesis of the DTMA ligand of the H-cluster. In addition, we show that ammonia is the source of the bridgehead nitrogen of DTMA.