A Redox Active [2Fe-2S] Cluster on the Hydrogenase Maturase HydF

Abstract

[FeFe]-hydrogenases are nature’s most prolific hydrogen catalysts, excelling at facilely interconverting H2 and protons. The catalytic core common to all [FeFe]-hydrogenases is a complex metallocofactor, referred to as the H-cluster, which is composed of a standard [4Fe-4S] cluster linked through a bridging thiolate to a 2Fe subcluster harboring dithiomethylamine, carbon monoxide, and cyanide ligands. This 2Fe subcluster is synthesized and inserted into [FeFe]-hydrogenase by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG are radical S-adenosylmethionine enzymes and synthesize the nonprotein ligands of the H-cluster. HydF is a GTPase that functions as a scaffold or carrier for 2Fe subcluster production. Herein, we utilize UV–visible, circular dichroism, and electron paramagnetic resonance spectroscopic studies to establish the existence of redox active [4Fe-4S] and [2Fe-2S] clusters bound to HydF. We have used spectroelectrochemical titrations to assign iron-sulfur cluster midpoint potentials, have shown that HydF purifies with a reduced [2Fe-2S] cluster in the absence of exogenous reducing agents, and have tracked iron–sulfur cluster spectroscopic changes with quaternary structural perturbations. Our results provide an important foundation for understanding the maturation process by defining the iron-sulfur cluster content of HydF prior to its interaction with HydE and HydG. We speculate that the [2Fe-2S] cluster of HydF either acts as a placeholder for HydG-derived Fe(CO)2CN species or serves as a scaffold for 2Fe subcluster assembly.

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

“…62 Additionally, this [4Fe-4S] cluster was the only Fe-S center revealed in this HydF structure. Specically no additional [2Fe-2S] cluster was found as suggested by prior spectroscopic studies, 29,63 and Caserta et al 62 suggest the prior evidence for such was obtained in HydF samples with incompletely loaded and degraded Fe-S clusters.…”

Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation

“…43,47 The genes for C.a. hydE , hydF (containing an N-terminal six-histidine tag), and hydG were cloned into pETDuet, pRSFDuet, and pCDFDuet vectors, respectively.…”

Overview of the Maturation Machinery of the H-Cluster of [FeFe]-Hydrogenases with a Focus on HydF

“…In addition to the [4Fe-4S] cluster, there is evidence from EPR spectroscopy that as-isolated and chemically reconstituted HydF can also host a [2Fe-2S] cluster [ 61 , 62 , 63 , 64 , 65 ], which is otherwise lacking in all solved HydF crystal structures [ 48 , 56 ]. Given this discrepancy, it is still unclear whether the presence of a [2Fe-2S] cluster represents a physiological state, a state of partial cluster maturation, or simply arises from an incomplete reconstitution due to the in vitro conditions.…”

“…The quaternary structure of the tetramer is uncertain since in the three available crystal structures the tetrameric assembly differs greatly and it is likely influenced by the crystal packing [ 56 ]. It is possible to separate the two forms in the recombinant protein via chromatographic methods, but the dimer and tetramer are in dynamic equilibrium [ 64 ]. A regulatory role has been attributed to the tetrameric form of HydF, acting as a switched-off state of the enzyme, since the tetrameric fractions are much less active in assembling the cluster [ 62 ].…”