Abstract
[FeFe]-hydrogenases catalyse the reduction of protons to hydrogen at a complex 2Fe[4Fe4S] center called H-cluster. The assembly of this active site is a multistep process involving three proteins, HydE, HydF and HydG. According to the current models, HydF has the key double role of scaffold, upon which the final H-cluster precursor is assembled, and carrier to transfer it to the target hydrogenase. The X-ray structure of HydF indicates that the protein is a homodimer with both monomers carrying two functional domains: a C-terminal FeS cluster-binding domain, where the precursor is assembled, and a N-terminal GTPase domain, whose exact contribution to cluster biogenesis and hydrogenase activation is still elusive. We previously obtained several hints suggesting that the binding of GTP to HydF could be involved in the interactions of this scaffold protein with the other maturases and with the hydrogenase itself. In this work, by means of site directed spin labeling coupled to EPR/PELDOR spectroscopy, we explored the conformational changes induced in a recombinant HydF protein by GTP binding, and provide the first clue that the HydF GTPase domain could be involved in the H-cluster assembly working as a molecular switch similarly to other known small GTPases.
Highlights
Biohydrogen production, one of the most promising frontiers in the field of renewable energies, is achieved in nature by several prokaryotic and eukaryotic microorganisms through a general class of evolutionarily unrelated metalloenzymes called hydrogenases
Since the presence of the other two domains may be important in producing structural constrains in HydF, by directing and/or amplifying the conformational changes induced at the GTP binding site, in the present work we used CW-electron paramagnetic resonance (EPR) and Pulse ELectron DOuble resonance (PELDOR) analysis by mapping the GTP-induced conformational changes along the entire HydF protein
According to previous experimental evidences indicating that the HydF GTPase activity is increased in the presence of K+19, the region of HydF nucleotide-binding G1 motif (...GRRNVGKSSFMNALV...) contains two asparagine residues, namely Asn[19] and Asn[27], which are highly conserved in the K+ activated G-proteins[37], as indicated by a detail of the multiple sequence alignment reported in panel C of Fig. 1
Summary
Biohydrogen production, one of the most promising frontiers in the field of renewable energies, is achieved in nature by several prokaryotic and eukaryotic microorganisms through a general class of evolutionarily unrelated metalloenzymes called hydrogenases. Three conserved proteins are involved in its biosynthesis and delivery, i.e. HydE, HydF and HydG, discovered in the unicellular green alga Chlamydomonas reinhardtii and found in all microorganisms containing a [FeFe]-hydrogenase[6] Both HydE and HydG are radical S-adenosylmethionine (SAM) proteins whereas HydF is a GTPase carrying a [4Fe4S] cluster binding motif[6]. The crystal structure of the apo-HydF protein showed the existence of flexible loops in this domain, which could undergo structural rearrangements upon GTP binding[8] This could in turn have an impact on the capability of the holo-protein to interact with HydE and HydG in the maturation machinery, and to drive the proper delivery of the H-cluster precursor to the target hydrogenase. We recognized sw[1] and sw[2] regions in the HydF GTPase domain and showed that, upon GTP binding, the protein undergoes conformational changes which are likely instrumental in promoting HydF activity in the maturation process of hydrogenases[35, 36]
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