title: Biochemical analysis of the interactions between the proteins involved in the [FeFe]-hydrogenase maturation process.
abstract: [FeFe]-hydrogenases are iron-sulfur proteins characterized by a complex active site, the H-cluster, whose assembly requires three conserved maturases. HydE and HydG are radical S-adenosylmethionine enzymes that chemically modify a H-cluster precursor on HydF, a GTPase with a dual role of scaffold on which this precursor is synthesized, and carrier to transfer it to the hydrogenase. Coordinate structural and functional relationships between HydF and the two other maturases are crucial for the H-cluster assembly. However, to date only qualitative analysis of this protein network have been provided. In this work we showed that the interactions of HydE and HydG with HydF are distinct events, likely occurring in a precise functional order driven by different kinetic properties, independently of the HydF GTPase activity, which is instead involved in the dissociation of the maturases from the scaffold. We also found that HydF is able to interact with the hydrogenase only when co-expressed with the two other maturases, indicating that under these conditions it harbors per se all the structural elements needed to transfer the H-cluster precursor, thus completing the maturation process. These results open new working perspectives aimed at improving the knowledge of how these complex metalloenzymes are biosynthesized.
authors.affiliation: Department of Biology, University of Padova, Viale G. Colombo 3, 35131 Padua, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Vallese
authors.forename: Paola
authors.initials: P
authors.lastname: Berto
authors.forename: Maria
authors.initials: M
authors.lastname: Ruzzene
authors.forename: Laura
authors.initials: L
authors.lastname: Cendron
authors.forename: Stefania
authors.initials: S
authors.lastname: Sarno
authors.forename: Edith
authors.initials: E
authors.lastname: De Rosa
authors.forename: Giorgio M
authors.initials: GM
authors.lastname: Giacometti
authors.forename: Paola
authors.initials: P
authors.lastname: Costantini
mesh_terms: D046969:Clostridium acetobutylicum / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006864:Hydrogenase / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D008667:Metalloproteins / Q000096:biosynthesis* / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D008667:Metalloproteins; D015534:Trans-Activators; C423908:zraR protein, E coli; D006864:Hydrogenase