Compositional and structural insights into the nature of the H-cluster precursor on HydF

Abstract

Assembly of an active [FeFe]-hydrogenase requires dedicated maturation enzymes that generate the active-site H-cluster: the radical SAM enzymes HydE and HydG synthesize the unusual non-protein ligands - carbon monoxide, cyanide, and dithiomethylamine - while the GTPase HydF serves as a scaffold for assembly of the 2Fe subcluster containing these ligands. In the current study, enzymatically cluster-loaded HydF ([2Fe]F) is produced by co-expression with HydE and HydG in an Escherichia coli host followed by isolation and examination by FTIR and EPR spectroscopy. FTIR reveals the presence of well-defined terminal CO and CN- ligands; however, unlike in the [FeFe]-hydrogenase, no bridging CO is observed. Exposure of this loaded HydF to exogenous CO or H2 produces no significant changes to the FTIR spectrum, indicating that, unlike in the [FeFe]-hydrogenase, the 2Fe cluster in loaded HydF is coordinatively saturated and relatively unreactive. EPR spectroscopy reveals the presence of both [4Fe-4S] and [2Fe-2S] clusters on this loaded HydF, but provides no direct evidence for these being linked to the [2Fe]F. Using the chemical reactivity and FTIR data, a large collection of computational models were evaluated. Their scaled quantum chemical vibrational spectra allowed us to score various [2Fe]F structures in terms of their ability to reproduce the diatomic stretching frequencies observed in the FTIR experimental spectra. Collectively, the results provide new insights that support the presence of a diamagnetic, but spin-polarized FeI-FeI oxidation state for the [2Fe]F precursor cluster that is coordinated by 4 CO and 2 CN- ligands, and bridged to an adjacent iron-sulfur cluster through one of the CN- ligands.

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

“…More specifically, HydE and HydG are radical SAM enzymes responsible for the construction of a [2Fe] subsite precatalyst on HydF, a scaffold protein that delivers the cofactor to the hydrogenase ( 7 – 17 ). A combination of FTIR spectroscopy, biochemical assays, biomimetic model chemistry, and DFT calculations has revealed that this precatalyst is strikingly similar to the synthetic [2Fe] subsite mimic [Fe 2 (adt)(CO) 4 (CN) 2 ] 2− ([2Fe] adt ) ( 14 , 18 – 20 ).…”

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.…”

“…In a recent work [ 65 ], the precursor to the [2Fe] H cluster present in HydF (called [2Fe] F cluster) has been suggested to be structurally different from the one present in HydA, based on FT-IR data (see Figure 1 for its structure). [2Fe] F has not been detected by EPR spectroscopy suggesting it is diamagnetic.…”

[FeFe]-hydrogenase maturation: H-cluster assembly intermediates tracked by electron paramagnetic resonance, infrared, and X-ray absorption spectroscopy

“…(adt)(CO) 4 (CN) 2 ] 2− ([2Fe] adt , Fig. 1c) [6,[27][28][29]. Indeed, incorporating the [2Fe] adt complex into either HydF or apo-HydA1 results in semi-synthetic forms of the two proteins, which are spectroscopically and biochemically indistinguishable from the native biological proteins [6,15,28,29,31].…”

“…A combination of biomimetic synthetic chemistry, biochemistry, spectroscopy, and quantum chemical calculations has revealed that the pre-catalyst on HydF is strikingly similar, if not identical, to the synthetic complex [Fe 2 I,I (adt)(CO) 4 (CN) 2 ] 2− ([2Fe] adt , Fig. 1 c) [ 6 , 27 – 29 ]. Indeed, incorporating the [2Fe] adt complex into either HydF or apo-HydA1 results in semi-synthetic forms of the two proteins, which are spectroscopically and biochemically indistinguishable from the native biological proteins [ 6 , 15 , 28 , 29 , 31 ].…”