Spectroscopic and saturation magnetization properties of the manganese- and cobalt-substituted Fur (ferric uptake regulation) protein from Escherichia coli.
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.
D001059:Apoproteins / Q000737:chemistry / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D001665:Binding Sites; D003035:Cobalt / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000737:chemistry*; D008024:Ligands; D008280:Magnetics; D008345:Manganese / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry* / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism