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, a and b 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.

Cysteine Mutational Studies Provide Insight into a Thiol-Based Redox Switch Mechanism of Metal and DNA Binding in FurA fromAnabaenasp. PCC 7120

“…FurA is a multifaceted protein that controls iron homeostasis and modulates an extensive regulon involved in a variety of cell functions (13,14,15). It also fulfills the features of a heme sensor protein whose interaction with this cofactor negatively affects its DNA binding ability in vitro (23,41).…”

“…On the other hand, FurA reductase activity occurs depending on the redox state of cysteines present in its CXXC motifs (5). Finally, the FurA-heme interaction that involves residue C 141 from one of the CXXC motifs undergoes redox-mediated ligand switching (41). Therefore, considering the apparent dependence of FurA activity on its oxidation state and the lack of a structural metal involving its two CXXC motifs, it can be envisaged that at least one redox-active cysteine would be required for the function of this protein.…”

“…These cysteines form a redox-sensitive disulfide bridge in a triple C101/ 104/133S mutant containing C 141 and C 144 (5). C 141 is also involved in Anabaena FurA-heme coordination (41).…”

Cj1386, an Atypical Hemin-Binding Protein, Mediates Hemin Trafficking to KatA in Campylobacter jejuni

“…Therefore, to determine residues involved in hemin binding, the Cj1386 protein sequence was aligned against Cj1386 homologs from different bacterial species to identify conserved residues that potentially were important for hemin binding. Heme coordination with proteins commonly occurs through histidine, tyrosine, methionine, or cysteine residues (22). A multiple-sequence alignment of Cj1386 against homologs of Campylobacter coli CCO11496, Helicobacter hepaticus YahD, Arcobacter butzleri Abu0197, and Pseudomonas putida Pp1834 is shown in Fig.…”

Unraveling the Redox Properties of the Global Regulator FurA fromAnabaenasp. PCC 7120: Disulfide Reductase Activity Based on Its CXXC Motifs

“…FurA also participates in the control of several genes involved in nitrogen metabolism, like the master regulator NtcA (41,42), and recent studies point to this protein as a heme-sensor in cyanobacteria (49). Phenotypic characterization of a FurA-overexpressing Anabaena strain, as well as the high representation of genes related to redox homeostasis as part of the FurA regulon (20,21), indicates that, in addition to controlling iron metabolism, this regulator is critical for cyanobacterial defense against oxidative injuries.…”