Maturation and assembly of [NiFe]-hydrogenases in Escherichia coli

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Hydrogenases are complex metalloenzymes that catalyse the reversible oxidation of molecular hydrogen. They can be sub-divided into three classes depending on the metal composition of their active sites. The actives sites of [NiFe]- and [FeFe]-hydrogenases share the unusual feature of having the diatomic ligands CN- and CO associated with the Fe atoms. Major advances have been made in studying the biosynthetic machinery involved in synthesis and attachment of these ligands to the iron atom in [NiFe]-hydrogenases, in particular using hydrogenase 3 of the hydrogen-evolving multi-protein formate hydrogenlyase complex of Escherichia coli as a model system. It has been clearly established that the CN- ligand, but not the CO, is derived from carbamoylphosphate. The metabolic route of CO delivery on the maturation pathway is not known, nor is anything known about the path of delivery of iron to the active site of these enzymes. Moreover, we understand little about the latter stages of hydrogenase assembly into a functional multi-protein complex. The aims of this research project are therefore to establish the metabolic route leading to CO ligand synthesis and attachment to the Fe atom in the active site of the [NiFe]-hydrogenases in E. coli, to determine the route of delivery of the iron atom to the active site and to determine how the formate hydrogenlyase complex is assembled.