Sirohaem is a cofactor of nitrite and sulfite reductases, essential for assimilation of nitrogen and sulfur. Sirohaem is synthesized from the central tetrapyrrole intermediate uroporphyrinogen III by methylation, oxidation and ferrochelation reactions. In Arabidopsis thaliana, the ferrochelation step is catalysed by sirohydrochlorin ferrochelatase (SirB), which, unlike its counterparts in bacteria, contains an [Fe–S] cluster. We determined the cluster to be a [4Fe–4S] type, which quickly oxidizes to a [2Fe–2S] form in the presence of oxygen. We also identified the cluster ligands as four conserved cysteine residues located at the C-terminus. A fifth conserved cysteine residue, Cys135, is not involved in ligating the cluster directly, but influences the oxygen-sensitivity of the [4Fe–4S] form, and possibly the affinity for the substrate metal. Substitution mutants of the enzyme lacking the Fe–S cluster or Cys135 retain the same specific activity in vitro and dimeric quaternary structure as the wild-type enzyme. The mutant variants also rescue a defined Escherichia coli sirohaem-deficient mutant. However, the mutant enzymes cannot complement Arabidopsis plants with a null AtSirB mutation, which exhibits post-germination arrest. These observations suggest an important physiological role for the Fe–S cluster in planta, highlighting the close association of iron, sulfur and tetrapyrrole metabolism.
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Research Article|
May 11 2012
Characterization of the evolutionarily conserved iron–sulfur cluster of sirohydrochlorin ferrochelatase from Arabidopsis thaliana
Kaushik Saha;
Kaushik Saha
1
*Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, U.K.
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Michael E. Webb;
Michael E. Webb
2
*Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, U.K.
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Stephen E. J. Rigby;
Stephen E. J. Rigby
†Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.
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Helen K. Leech;
Helen K. Leech
3
‡School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, U.K.
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Martin J. Warren;
Martin J. Warren
‡School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, U.K.
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Alison G. Smith
Alison G. Smith
4
*Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, U.K.
4To whom correspondence should be addressed (email as25@cam.ac.uk).
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Publisher: Portland Press Ltd
Received:
November 23 2011
Revision Received:
March 12 2012
Accepted:
March 13 2012
Accepted Manuscript online:
March 13 2012
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2012 Biochemical Society
2012
Biochem J (2012) 444 (2): 227–237.
Article history
Received:
November 23 2011
Revision Received:
March 12 2012
Accepted:
March 13 2012
Accepted Manuscript online:
March 13 2012
Citation
Kaushik Saha, Michael E. Webb, Stephen E. J. Rigby, Helen K. Leech, Martin J. Warren, Alison G. Smith; Characterization of the evolutionarily conserved iron–sulfur cluster of sirohydrochlorin ferrochelatase from Arabidopsis thaliana. Biochem J 1 June 2012; 444 (2): 227–237. doi: https://doi.org/10.1042/BJ20111993
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