Insights into the role of HydF-catalyzed GTP activity in hydrogenase maturation
Bueling, Alexandra Lee.
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The [FeFe]-hydrogenase contains an unusual active-site cluster, the H-cluster, composed of a [4Fe-4S] cluster bridged to a 2Fe subunit coordinated by CO, CN-, and a nonprotein bridging dithiolate. Three accessory proteins, HydE, HydF, and HydG are required for maturation of the H-cluster. It has been proposed that the GTP-hydrolyzing protein HydF acts as a scaffold for assembly of the [2Fe] subcluster, which is then transferred to hydrogenase to produce the active enzyme[McGlynn, S.M.; Shepard, E.M.; Winslow, M.A.; Naumov, A.V.; Duschene, K.S.; Posewitz, M.C.; Broderick, W.E.; Broderick, J.B.; Peters, J.W. FEBS Lett. 2008, 582, 2183.]. The precise roles of HydE, HydF, and HydG in this process, however, remain unclear. The overall goal of this work is to study the role of HydF in hydrogenase maturation. In vivo studies have identified specific amino acid residues of HydF necessary for production of active hydrogenase [King, P.W.; Posewitz, M.C.; Ghirardi, M.L.; Seibert, M. J Bacteriol. 2006, 188, 2163.]. The objectives of this study were to: 1) investigate the involvement of each of these amino acid residues on the GTP hydrolyzing ability of HydF 2) examine the effects of structural and accessory hydrogenase proteins on rate of GTP hydrolysis 3) examine the importance of [Fe-S] cluster in HydF GTPase activity. GTPase assays were analyzed by high performance liquid chromatography (HPLC). The results indicate mutations to conserved glycine residues in the GTP binding loop, and a potential iron ligand for the [Fe-S] cluster are deleterious to GTPase activity. Another mutation in the GTP binding loop involved in Mg ²+ coordination appears to have little effect on the rate of GTP hydrolysis. The two accessory proteins HydE and HydG increased GTPase activity, while the hydrogenase structural protein (HydA) appears to have little effect, supporting the suggestion that HydF acts as a scaffold. The presence or absence of an iron-sulfur cluster does not significantly affect GTPase activity. Together, these results suggest GTP hydrolysis does not play a role in transfer of activating elements to hydrogenase, but does play a role in hydrogenase maturation.