Compositional and structural insights into the nature of the H-cluster precursor on HydF†,Dalton Transactions

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Compositional and structural insights into the nature of the H-cluster precursor on HydF†
Dalton Transactions ( IF 4 ) Pub Date : 2018-06-19 00:00:00 , DOI: 10.1039/c8dt01654b
Anna G. Scott _{1,

2,

3,

4} , Robert K. Szilagyi _{1,

2,

3,

4} , David W. Mulder _{4,

5,

6,

7} , Michael W. Ratzloff _{4,

5,

6,

7} , Amanda S. Byer _{1,

2,

3,

4} , Paul W. King _{4,

5,

6,

7} , William E. Broderick _{1,

2,

3,

4} , Eric M. Shepard _{1,

2,

3,

4} , Joan B. Broderick _{1,

2,

3,

4}

Affiliation

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 Fe^I–Fe^I 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.

更新日期：2018-06-19

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