EPR spectroscopic studies of the Fe-S clusters in the O2- tolerant [NiFe]-hydrogenase Hyd-1 from Escherichia coli and characterization of the unique [4Fe-3S] cluster by HYSCORE

Roessler, Maxie M., Evans, Rhiannon M., Davies, Rosalind A., Harmer, Jeffrey and Armstrong, Fraser A. (2012) EPR spectroscopic studies of the Fe-S clusters in the O2- tolerant [NiFe]-hydrogenase Hyd-1 from Escherichia coli and characterization of the unique [4Fe-3S] cluster by HYSCORE. Journal of the American Chemical Society, 134 37: 15581-15594. doi:10.1021/ja307117y


Author Roessler, Maxie M.
Evans, Rhiannon M.
Davies, Rosalind A.
Harmer, Jeffrey
Armstrong, Fraser A.
Title EPR spectroscopic studies of the Fe-S clusters in the O2- tolerant [NiFe]-hydrogenase Hyd-1 from Escherichia coli and characterization of the unique [4Fe-3S] cluster by HYSCORE
Formatted title
EPR spectroscopic studies of the Fe-S clusters in the O2- tolerant [NiFe]-hydrogenase Hyd-1 from Escherichia coli and characterization of the unique [4Fe-3S] cluster by HYSCORE
Journal name Journal of the American Chemical Society   Check publisher's open access policy
ISSN 0002-7863
1520-5126
Publication date 2012-09
Sub-type Article (original research)
DOI 10.1021/ja307117y
Volume 134
Issue 37
Start page 15581
End page 15594
Total pages 14
Place of publication Washington, United States
Publisher American Chemical Society
Language eng
Formatted abstract
The unusual [4Fe–3S] cluster proximal to the active site plays a crucial role in allowing a class of [NiFe]-hydrogenases to function in the presence of O2 through its unique ability to undergo two rapid, consecutive one-electron transfers. This property helps to neutralize reactive oxygen species. Mechanistic details and the role of the medial and distal clusters remain unresolved. To probe the Fe–S relay, continuous wave and pulse electron paramagnetic resonance (EPR) studies were conducted on the O2-tolerant hydrogenase from Escherichia coli (Hyd-1) and three variants with point mutations at the proximal and/or medial clusters. Reduction potentials of the proximal ([4Fe–3S]5+/4+/3+) and medial ([3Fe–4S]+/0) clusters were determined by potentiometry. The medial [3Fe–4S]+/0 reduction potential is exceptionally high, implicating a mechanistic role in O2-tolerance. Numerous experiments establish that the distal cluster has a ground state S > 1/2 in all three variants and indicate that this is also the case for native Hyd-1. Concurrent with the Hyd-1 crystal structure, EPR data for the ‘superoxidized’ P242C variant, in which the medial cluster is ‘magnetically silenced’, reveal two conformations of the proximal [4Fe–3S]5+ cluster, and X-band HYSCORE spectroscopy shows two 14N hyperfine couplings attributed to one conformer. The largest, A(14N) = [11.5,11.5,16.0] ± 1.5 MHz, characterizes the unusual bond between one Fe (Fe4) and the backbone amide-N of cysteine-20. The second, A(14N) = [2.8,4.6,3.5] ± 0.3 MHz, is assigned to NC19. The 14N hyperfine couplings are conclusive evidence that Fe4 is a valence-localized Fe3+ in the superoxidized state, whose formation permits an additional electron to be transferred rapidly back to the active site during O2 attack.
Keyword Alternative spin states
Iron sulfur clusters
Desulfovibrio gigas hydrogenase
Pyrococcus furiosus ferredoxin
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: Centre for Advanced Imaging Publications
 
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