title: Characterization of the evolutionarily conserved iron-sulfur cluster of sirohydrochlorin ferrochelatase from Arabidopsis thaliana.
abstract: 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, Cys(135), 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 Cys(135) 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.
authors.affiliation: Department of Plant Sciences, University of Cambridge, UK.
authors.forename: Kaushik
authors.initials: K
authors.lastname: Saha
authors.forename: Michael E
authors.initials: ME
authors.lastname: Webb
authors.forename: Stephen E J
authors.initials: SE
authors.lastname: Rigby
authors.forename: Helen K
authors.initials: HK
authors.lastname: Leech
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Warren
authors.forename: Alison G
authors.initials: AG
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D017124:Conserved Sequence; D019143:Evolution, Molecular; D005294:Ferrochelatase / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010936:Plant Extracts / Q000737:chemistry / Q000235:genetics; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D014578:Uroporphyrins / Q000737:chemistry* / Q000235:genetics
chemical_list: D007506:Iron-Sulfur Proteins; D010936:Plant Extracts; D014578:Uroporphyrins; C014188:sirohydrochlorin; D005294:Ferrochelatase