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

Abstract

Nature utilizes [FeFe]-hydrogenase enzymes to catalyze the interconversion between H2 and protons and electrons. Catalysis occurs at the H-cluster, a carbon monoxide-, cyanide-, and dithiomethylamine-coordinated 2Fe subcluster bridged via a cysteine to a [4Fe-4S] cluster. Biosynthesis of this unique metallocofactor is accomplished by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG belong to the radical S-adenosylmethionine superfamily of enzymes and synthesize the nonprotein ligands of the H-cluster. These enzymes interact with HydF, a GTPase that acts as a scaffold or carrier protein during 2Fe subcluster assembly. Prior characterization of HydF demonstrated the protein exists in both dimeric and tetrameric states and coordinates both [4Fe-4S]2+/+ and [2Fe-2S]2+/+ clusters [Shepard, E. M., Byer, A. S., Betz, J. N., Peters, J. W., and Broderick, J. B. (2016) Biochemistry 55, 3514–3527]. Herein, electron paramagnetic resonance (EPR) is utilized to characterize the [2Fe-2S]+ and [4Fe-4S]+ clusters bound to HydF. Examination of spin relaxation times using pulsed EPR in HydF samples exhibiting both [4Fe-4S]+ and [2Fe-2S]+ cluster EPR signals supports a model in which the two cluster types either are bound to widely separated sites on HydF or are not simultaneously bound to a single HydF species. Gel filtration chromatographic analyses of HydF spectroscopic samples strongly suggest the [2Fe-2S]+ and [4Fe-4S]+ clusters are coordinated to the dimeric form of the protein. Lastly, we examined the 2Fe subcluster-loaded form of HydF and showed the dimeric state is responsible for [FeFe]-hydrogenase activation. Together, the results indicate a specific role for the HydF dimer in the H-cluster biosynthesis pathway.

Iron–Sulfur Cluster States of the Hydrogenase Maturase HydF

“…Examining the biosynthetic role of this HydF, we show that dimeric HydF EG productively interacts with HydA ΔEFG and transfers the [2Fe] H cluster to afford HydA activation. 5 Together, the observations indicate a specific role for the HydF dimer in the H-cluster biosynthetic pathway.…”

“…Insight into these issues was gained via the analysis of spin relaxation times using pulsed EPR in HydF Δ EG samples exhibiting both [4Fe-4S] + and [2Fe-2S] + cluster EPR signals. 5 Simulations of relaxation rates established that the HydF ΔEG [2Fe-2S] + cluster is not near, or directly bridged to, the [4Fe-4S] + cluster. As these HydF Δ EG samples were predominantly dimeric in nature, this suggests that the [2Fe-2S] + and [4Fe-4S] + clusters are coordinated within different monomeric subunits of the dimer at a distance of ≥ 25 Å, or one population of dimeric HydF ΔEG contains only the [2Fe-2S] + cluster while another population of the dimeric enzyme contains only the [4Fe-4S] + cluster.…”

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.…”

“…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. Since there are no other binding motifs in HydF, and its relaxation properties suggest that the [2Fe-2S] cluster is located at least 2.5 nm away from the main cluster, it is likely bound in the same binding site in a different monomer, with a mixed occupancy of the sites in the two monomers, or a subset of proteins binding only a single type of cluster [ 62 ]. Finally, while there is a consensus about the role of the [4Fe-4S] cluster as the anchor for the [2Fe] H cluster, the role of [2Fe-2S] cluster is completely undefined.…”

“…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 ].…”