Periplasmic SER (selenate reductase) from Thauera selenatis is classified as a member of the Tat (twin-arginine translocase)-translocated (Type II) molybdoenzymes and comprises three subunits each containing redox cofactors. Variable-temperature X-band EPR spectra of the purified SER complex showed features attributable to centres [3Fe–4S]1+, [4Fe–4S]1+, Mo(V) and haem-b. EPR-monitored redox-potentiometric titration of the SerABC complex (SerA–SerB–SerC, a hetero-trimetric complex of αβγ subunits) revealed that the [3Fe–4S] cluster (FS4, iron-sulfur cluster 4) titrated as n=1 Nernstian component with a midpoint redox potential (Em) of +118±10 mV for the [3Fe–4S]1+/0 couple. A [4Fe–4S]1+ cluster EPR signal developed over a range of potentials between 300 and −200 mV and was best fitted to two sequential Nernstian n=1 curves with midpoint redox potentials of +183±10 mV (FS1) and −51±10 mV (FS3) for the two [4Fe–4S]1+/2+ cluster couples. Upon further reduction, the observed signal intensity of the [4Fe–4S]1+ cluster decreases. This change in intensity can again be fitted to an n=1 Nernstian component with a midpoint potential (Em) of about −356 mV (FS2). It is considered likely that, at low redox potential (Em less than −300 mV), the remaining oxidized cluster is reduced (spin S=1/2) and strongly spin-couples to a neighbouring [4Fe–4S]1+ cluster rendering both centres EPR-silent. The involvement of both [3Fe–4S] and [4Fe–4S] clusters in electron transfer to the active site of the periplasmic SER was demonstrated by the re-oxidation of the clusters under anaerobic selenate turnover conditions. Attempts to detect a high-spin [4Fe–4S] cluster (FS0) in SerA at low temperature (5 K) and high power (100 mW) were unsuccessful. The Mo(V) EPR recorded at 60 K, in samples poised at pH 6.0, displays principal g values of g3∼1.999, g2∼1.996 and g1∼1.965 (gav 1.9867). The dominant features at g2 and g3 are not split, but hyperfine splitting is observed in the g1 region of the spectrum and can be best simulated as arising from a single proton with a coupling constant of A1 (1H)=1.014 mT. The presence of the haem-b moiety in SerC was demonstrated by the detection of a signal at g∼3.33 and is consistent with haem co-ordinated by methionine and lysine axial ligands. The combined evidence from EPR analysis and sequence alignments supports the assignment of the periplasmic SER as a member of the Type II molybdoenzymes and provides the first spectro-potentiometric insight into an enzyme that catalyses a key reductive reaction in the biogeochemical selenium cycle.
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Research Article|
October 29 2007
Investigation of the redox centres of periplasmic selenate reductase from Thauera selenatis by EPR spectroscopy
Elizabeth J. Dridge;
Elizabeth J. Dridge
1
*School of Biosciences, Centre for Biocatalysis, University of Exeter, Stocker Road, Exeter EX4 4QD, U.K.
†Institute for Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne NE2 4HH, U.K.
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Carys A. Watts;
Carys A. Watts
1
†Institute for Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne NE2 4HH, U.K.
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Brian J. N. Jepson;
Brian J. N. Jepson
‡School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.
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Kirsty Line;
Kirsty Line
*School of Biosciences, Centre for Biocatalysis, University of Exeter, Stocker Road, Exeter EX4 4QD, U.K.
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Joanne M. Santini;
Joanne M. Santini
§Department of Biology, University College London, Gower Street, London WC1E 6BT, U.K.
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David J. Richardson;
David J. Richardson
‡School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.
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Clive S. Butler
Clive S. Butler
2
*School of Biosciences, Centre for Biocatalysis, University of Exeter, Stocker Road, Exeter EX4 4QD, U.K.
2To whom correspondence should be addressed (email c.s.butler@exeter.ac.uk).
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Publisher: Portland Press Ltd
Received:
May 18 2007
Revision Received:
July 20 2007
Accepted:
August 10 2007
Accepted Manuscript online:
August 10 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2007 Biochemical Society
2007
Biochem J (2007) 408 (1): 19–28.
Article history
Received:
May 18 2007
Revision Received:
July 20 2007
Accepted:
August 10 2007
Accepted Manuscript online:
August 10 2007
Citation
Elizabeth J. Dridge, Carys A. Watts, Brian J. N. Jepson, Kirsty Line, Joanne M. Santini, David J. Richardson, Clive S. Butler; Investigation of the redox centres of periplasmic selenate reductase from Thauera selenatis by EPR spectroscopy. Biochem J 15 November 2007; 408 (1): 19–28. doi: https://doi.org/10.1042/BJ20070669
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