Structure of Hydrogenase Maturation Protein HypF with Reaction Intermediates Shows Two Active Sites

Abstract

[NiFe]-hydrogenases are multimeric proteins. Thelarge subunit contains the NiFe(CN)(2)CO bimetallic active center and the small subunit contains Fe-S clusters. Biosynthesis and assembly of the NiFe(CN)(2)CO active center requires six Hyp accessory proteins. The synthesis of the CN(-) ligands iscatalyzed by the combined actions of HypF andHypE using carbamoylphosphate as a substrate.We report the structure of Escherichia coli HypF(92-750) lacking the N-terminal acylphosphatase domain. HypF(92-750) comprises the novel Zn-finger domain, the nucleotide-binding YrdC-like domain, and the Kae1-like universal domain, also binding a nucleotide and a Zn(2+) ion. The two nucleotide-binding sites are sequestered in an internal cavity, facing each other and separated by ∼14Å. The YrdC-like domain converts carbamoyl moiety to a carbamoyl adenylate intermediate, which is channeled to the Kae1-like domain. Mutations within either nucleotide-binding site compromise hydrogenase maturation but do not affect the carbamoylphosphate phosphatase activity.