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Issue 28, 2018
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Compositional and structural insights into the nature of the H-cluster precursor on HydF

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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.

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

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Publication details

The article was received on 25 Apr 2018, accepted on 19 Jun 2018 and first published on 19 Jun 2018


Article type: Paper
DOI: 10.1039/C8DT01654B
Dalton Trans., 2018,47, 9521-9535

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

    A. G. Scott, R. K. Szilagyi, D. W. Mulder, M. W. Ratzloff, A. S. Byer, P. W. King, W. E. Broderick, E. M. Shepard and J. B. Broderick, Dalton Trans., 2018, 47, 9521
    DOI: 10.1039/C8DT01654B

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