Spectroscopic and Saturation Magnetization Properties of the Manganese- and Cobalt-Substituted Fur (Ferric Uptake Regulation) Protein from Escherichia coli
详细信息 查看全文
- 作者：Annie Adrait ; Lilian Jacquamet ; Laurent Le Pape ; Anne Gonzalez de Peredo ; Daniel Aberdam ; Jean-Louis Hazemann ; Jean-Marc Latour ; and Isabelle Michaud-Soret
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：May 11, 1999
- 年：1999
- 卷：38
- 期：19
- 页码：6248 - 6260
- 全文大小：145K
- 年卷期：v.38,no.19(May 11, 1999)
- ISSN：1520-4995
文摘
The Fur apoprotein has been purified and reconstituted with Co2+ and Mn2+ ions. These sampleshave been analyzed by UV-visible, EPR, and 1H NMR spectroscopies, by XAS, and by magnetizationmeasurements. The apo-Fur protein is able to bind one metal dication (Co2+ or Mn2+) per monomer. Asaturation magnetization study confirms the presence of a high-spin metal dication [Mn(II) S = 5/2 andCo(II) S = 3/2]. The two metal ions per Fur dimer are not in magnetic interaction (
J < 0.1 cm-1 ). TheUV-visible spectrum of the cobalt-substituted form (Co-Fur) presents two main bands at 660 nm and540(br) nm with 
DER=0 >540 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 valueof 55 cm-1 for the splitting (
) between the ground and the first excited state has been derived from anEPR saturation study and is in agreement with magnetization data. The EXAFS data of Co-Fur indicatea metal environment comprising five nitrogen/oxygen atoms at 2.11 Å, the absence of sulfur, and thepresence of histidines as ligands. 1H NMR of Co-Fur in H2O and D2O shows at least two exchangeablesignals coming from histidine NH protons and shows the signature of carboxylate group(s). The combinedspectroscopic data allow us to propose that the main metal site of Fur in Co-Fur contains at least twohistidines, at least one aspartate or glutamate, and no cysteine as ligands and is in an axially distortedoctahedral environment.
J < 0.1 cm-1 ). TheUV-visible spectrum of the cobalt-substituted form (Co-Fur) presents two main bands at 660 nm and540(br) nm with DER=0 >540 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 valueof 55 cm-1 for the splitting () between the ground and the first excited state has been derived from anEPR saturation study and is in agreement with magnetization data. The EXAFS data of Co-Fur indicatea metal environment comprising five nitrogen/oxygen atoms at 2.11 Å, the absence of sulfur, and thepresence of histidines as ligands. 1H NMR of Co-Fur in H2O and D2O shows at least two exchangeablesignals coming from histidine NH protons and shows the signature of carboxylate group(s). The combinedspectroscopic data allow us to propose that the main metal site of Fur in Co-Fur contains at least twohistidines, at least one aspartate or glutamate, and no cysteine as ligands and is in an axially distortedoctahedral environment.