caa a22 4500 605545936 CHVBK 20210128100938.0 cr unu---uuuuu 210128e20150401xx s 000 0 eng 10.1007/s00723-015-0641-z doi (NATIONALLICENCE)springer-10.1007/s00723-015-0641-z A conformational study of the GTPase domain of [FeFe]-hydrogenase maturation protein HydF by PELDOR spectroscopy [Elektronische Daten] [Lorenzo Maso, Laura Galazzo, Francesca Vallese, Marilena Di Valentin, Marco Albertini, Edith De Rosa, Giorgio Giacometti, Paola Costantini, Donatella Carbonera] [FeFe]-hydrogenases catalyze the reversible interconversion of protons to molecular hydrogen (H2) at an active site called H-cluster. The maturation pathway of these enzymes is a complex process involving three proteins, HydE, HydF and HydG. The maturase protein HydF has been suggested to interact with HydE and HydG and to be the transferase that shuttles the complete H-cluster to the hydrogenase; however, the exact molecular mechanism driving this translocation remains unclear. HydF is constituted by three different domains: a N-terminal GTP-binding domain, a dimerization domain and a C-terminal [4Fe4S] cluster-binding domain. To investigate possible conformational changes induced by the GTP binding in the N-terminal domain, we have expressed, in Escherichia coli, a recombinant HydF protein from Thermotoga neapolitana including the GTP-binding domain only. Site-directed mutants were designed in which the native residues were substituted by cysteines and subsequently spin labeled with the nitroxide MTSSL. CW-EPR was used to study the local mobility of the nitroxides at each site, and double spin-labeled mutants have been investigated by PELDOR spectroscopy. We found that the binding of the nucleotide does not induce large conformational effects within the isolated GTP domain, at least at the level of the elements investigated in this work. However, small changes in the distance between spin labels were observed which might reflect diffuse structural rearrangements. We suggest that the variations following the GTP binding could affect the dimer form adopted by the whole HydF protein in solution and, as a consequence, the interactions with the other maturases. Springer-Verlag Wien, 2015 Maso Lorenzo Department of Biology, University of Padova, Viale G. Colombo 3, 35131, Padua, Italy aut Galazzo Laura Department of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131, Padua, Italy aut Vallese Francesca Department of Biomedical Sciences, University of Padova, Viale G. G. Colombo 3, 35131, Padua, Italy aut Di Valentin Marilena Department of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131, Padua, Italy aut Albertini Marco Department of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131, Padua, Italy aut De Rosa Edith Department of Biology, University of Padova, Viale G. Colombo 3, 35131, Padua, Italy aut Giacometti Giorgio Department of Biology, University of Padova, Viale G. Colombo 3, 35131, Padua, Italy aut Costantini Paola Department of Biology, University of Padova, Viale G. Colombo 3, 35131, Padua, Italy aut Carbonera Donatella Department of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131, Padua, Italy aut Applied Magnetic Resonance Springer Vienna 46/4(2015-04-01), 465-479 0937-9347 46:4<465 2015 46 723 https://doi.org/10.1007/s00723-015-0641-z text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00723-015-0641-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Maso Lorenzo Department of Biology, University of Padova, Viale G. Colombo 3, 35131, Padua, Italy aut NATIONALLICENCE 700 1- Galazzo Laura Department of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131, Padua, Italy aut NATIONALLICENCE 700 1- Vallese Francesca Department of Biomedical Sciences, University of Padova, Viale G. G. Colombo 3, 35131, Padua, Italy aut NATIONALLICENCE 700 1- Di Valentin Marilena Department of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131, Padua, Italy aut NATIONALLICENCE 700 1- Albertini Marco Department of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131, Padua, Italy aut NATIONALLICENCE 700 1- De Rosa Edith Department of Biology, University of Padova, Viale G. Colombo 3, 35131, Padua, Italy aut NATIONALLICENCE 700 1- Giacometti Giorgio Department of Biology, University of Padova, Viale G. Colombo 3, 35131, Padua, Italy aut NATIONALLICENCE 700 1- Costantini Paola Department of Biology, University of Padova, Viale G. Colombo 3, 35131, Padua, Italy aut NATIONALLICENCE 700 1- Carbonera Donatella Department of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131, Padua, Italy aut NATIONALLICENCE 773 0- Applied Magnetic Resonance Springer Vienna 46/4(2015-04-01), 465-479 0937-9347 46:4<465 2015 46 723