Spectroscopic and Saturation Magnetization Properties of the Manganese- and Cobalt-Substituted Fur (Ferric Uptake Regulation) Protein from Escherichia coli

Abstract

The Fur apoprotein has been purified and reconstituted with Co2+ and Mn2+ ions. These samples have been analyzed by UV-visible, EPR, and 1H NMR spectroscopies, by XAS, and by magnetization measurements. The apo-Fur protein is able to bind one metal dication (Co2+ or Mn2+) per monomer. A saturation magnetization study confirms the presence of a high-spin metal dication [Mn(II) S = 5/2 and Co(II) S = 3/2]. The two metal ions per Fur dimer are not in magnetic interaction (|J| < 0.1 cm-1 ). The UV-visible spectrum of the cobalt-substituted form (Co-Fur) presents two main bands at 660 nm and 540(br) nm with epsilon540 nm = 65 M-1 cm-1. The EPR spectrum gives the following g values: gx = 5.0(5), gy = 4.0(2), and gz = 2. 3(1), which are in accordance with a nearly axial (E/D < 0.11) site. The value of 55 cm-1 for the splitting (Delta) between the ground and the first excited state has been derived from an EPR saturation study and is in agreement with magnetization data. The EXAFS data of Co-Fur indicate a metal environment comprising five nitrogen/oxygen atoms at 2.11 A, the absence of sulfur, and the presence of histidines as ligands. 1H NMR of Co-Fur in H2O and D2O shows at least two exchangeable signals coming from histidine NH protons and shows the signature of carboxylate group(s). The combined spectroscopic data allow us to propose that the main metal site of Fur in Co-Fur contains at least two histidines, at least one aspartate or glutamate, and no cysteine as ligands and is in an axially distorted octahedral environment.

Direct inhibition by nitric oxide of the transcriptional ferric uptake regulation protein via nitrosylation of the iron

“…Recent data concerning the nature of the ligands coordinated to iron in FeFur are much more in agreement with the environments in these compounds. Indeed, spectroscopic data on cobalt-and iron-substituted Fur indicated that the metal is hexacoordinated with one ligand at a longer distance and only nitrogen and oxygen donor ligands including two (or three) histidines and one (or two) aspartate or glutamate (20,21). In conclusion, the nature of the EPR signal of the nitrosyl iron species in the FeFur-NO complex cannot be assigned without further spectroscopic characterizations.…”

“…Fur expression was induced at OD 600 ϭ 0.7-0.8 by isopropyl ␤-D-thiogalactoside (200 mM, 600 l) during 2 h and 30 min. Dimeric Fur protein was purified as described (20). The buffer was exchanged for 100 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane͞100 mM KCl, pH 7.5.…”

“…The purity was checked from SDS͞PAGE. Protein concentrations were calculated by using an absorption coefficient at 275 nm of 0.4 mg Ϫ1 ⅐ml⅐cm Ϫ1 for the monomer of pure apo-Fur (20).…”

“…Fur is the key protein for the control of the intracellular iron concentration. The active form of the Fur protein, FeFur, contains a nonheme ferrous iron site with oxygen and nitrogen donor ligands (20,21). When the cellular iron level becomes too low, the active Fur repressor looses Fe 2ϩ , its corepressor, and is no longer able to bind to specific DNA sequences.…”

The interaction between sulfathiazole and cobalt(II): potentiometric studies

“…Spectroscopic characterization of these systems has revealed that cobalt can be present as Co(II) and Co(III) ions in several types of coordination, which lead to different electronic and magnetic properties. In this context, the use of simple Cocontaining systems with low molecular weight ligands is useful for understanding the correlation between spectroscopic and structural properties [19][20][21][22] .…”