title: Site-directed mutagenesis and spectral studies suggest a putative role of FurA from Anabaena sp. PCC 7120 as a heme sensor protein.
abstract: The transcriptional repressor Fur (ferric uptake regulator) is one of the most important switches regulating prokaryotic iron metabolism. Cyanobacterial FurA binds heme in the micromolar concentration range and this interaction negatively affects its in vitro DNA binding ability in a concentration-dependent manner. Using site-directed mutagenesis along with difference absorption UV-visible, circular dichroism and electronic paramagnetic resonance spectroscopies, we further analyse the nature of heme binding in FurA. Our data point to Cys141, within a Cys-Pro motif, as an axial ligand of the Fe(III) high-spin heme. In the Fe(II) oxidation state, the heme shows low-spin form with an electronic absorption spectrum typical of six-coordinated low-spin heme proteins with a Soret absorption maximum blue-shifted by 25 nm in relation to typical low-spin thiolate-ligated Fe(II) heme proteins. Moreover, the ferrous C141S mutant shows Soret, α and β bands almost identical to those observed for ferrous wild-type heme-FurA, indicating that the heme in ferrous C141S is in the same six-coordinated heme ligation as the ferrous native form. Therefore, Cys141 is not a ligand of the Fe(II) heme centre, suggesting a redox-dependent ligand switch undergone by this regulator. Our results indicate that the binding of heme to FurA exhibits the same physicochemical features as previously described for heme sensor proteins.
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, and Institute of Biocomputation and Physics of Complex Systems (BiFi) - Associated Unit to IQRS-CSIC, University of Zaragoza, Spain.
authors.forename: Silvia
authors.initials: S
authors.lastname: Pellicer
authors.forename: Andrés
authors.initials: A
authors.lastname: González
authors.forename: M Luisa
authors.initials: ML
authors.lastname: Peleato
authors.forename: Jesús I
authors.initials: JI
authors.lastname: Martinez
authors.forename: María F
authors.initials: MF
authors.lastname: Fillat
authors.forename: M Teresa
authors.initials: MT
authors.lastname: Bes
mesh_terms: D017033:Anabaena / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D006418:Heme / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D013056:Spectrophotometry, Ultraviolet
chemical_list: D001426:Bacterial Proteins; D006418:Heme