The maturase HydF enables [FeFe] hydrogenase assembly via transient, cofactor-dependent interactions

Abstract

<jats:p>[FeFe] hydrogenases have attracted extensive attention in the field of renewable energy research due to their remarkable efficiency for H2 gas production. H2 formation is catalyzed by a biologically unique hexanuclear iron cofactor denoted the “H-cluster”. The assembly of this cofactor requires a dedicated maturation machinery including HydF, a multidomain [4Fe4S] cluster protein with GTPase activity. HydF is responsible for harboring and delivering a pre-catalyst to the apo-hydrogenase, but the details of this process are not well understood. Herein we utilize Gas-phase Electrophoretic MacroMolecule Analysis to show that a HydF dimer forms a transient interaction complex with the hydrogenase, and that the formation of this complex is dependent on the cofactor content on HydF. Moreover, FTIR, EPR and UV/Vis spectroscopy studies of mutants of HydF show that the isolated iron-sulfur cluster domain retains the capacity for binding the pre-catalyst in a reversible fashion, and is capable of activating apo-hydrogenase in in vitro assays. These results demonstrate the central role of the iron-sulfur cluster domain of HydF in final stages of H-cluster assembly, i.e. in binding and delivering the pre-catalyst.</jats:p>

The [4Fe‐4S]‐Cluster of HydF is not Required for the Binding and Transfer of the Diiron Site of [FeFe]‐Hydrogenases

“…In both cases would the signal shifts suggest a close proximity of [2Fe] MIM to the [4Fe‐4S]‐cluster niche, especially for the CN − ligand. This suggestion is supported by previous studies in which truncated forms of HydF from Thermotoga maritima that only contain the cluster binding domain are still capable of binding [2Fe] MIM [10c] . Moreover, a signal shift of the overall FTIR band pattern of Tme HydF WT ‐[2Fe] MIM was observed upon reduction with sodium dithionite (NaDT), but not when the C298S variant was reduced (Figure S5).…”

Biosynthesis of the catalytic H-cluster of [FeFe] hydrogenase: the roles of the Fe–S maturase proteins HydE, HydF, and HydG

“…Experiments are now revealing details of how this synthetic cluster, as installed in HydF, is transferred to HydA. 66,67 Of course such studies do not directly indicate how nature introduces the binuclear cluster when it is not preassembled by talented synthetic chemists. Is some binuclear precursor rst formed by HydE in the cavity adjacent to the HydE's SAM cluster, to then be transferred to HydF en route to HydA1 along parallel lines as observed for the synthetic binuclear cluster?…”