The histidines of the iron‐uptake regulation protein, Fur

Abstract

There are 12 histidine residues/molecule in the iron-uptake regulation protein (Fur). Here we examine their pH dependence using proton nuclear magnetic resonance spectroscopy. The histidines have widely spread acid dissociation constants but we can not offer a simple explanation for their complicated behaviour.The iron-uptake regulation protein, Fur, has 12 histidine residues/molecule and some of these have been implicated in iron binding [l -31. This iron binding is affected by pH and thus the Fur protein is not just a monitor of iron concentration but a combined monitor of iron concentration and pH as well. For this reason we have carried out a detailed study of the histidines of Fur using proton nuclear magnetic resonance ('H-NMR) spectroscopy to follow their behaviour on changing pH.There are many NMR studies of histidine resonances in proteins as a function of pH which we used to guide our work [4 ~ 61. The most sophisticated analysis treated the perturbation of a simple histidine in ribonuclease, including a consideration of neighbouring group effects, on both pKa and on the chemical shifts of the resonances both of the protonated and deprotonated histidine residues [7 -91. A parallel study of the isolated histidines of haemoglobin was used to analyse long-range interactions [lo, 111. Here we show that the behaviour of the histidine resonances of Fur is yet more complicated and we suggested that this could arise from cooperativity effects combined with pKa and chemical shift modulations. Our data are not open to very detailed analysis due to the complexity of the systems. MATERIALS AND METHODSThe Fur protein was provided by Prof. J. B. Neilands (Department of Biochemistry, University of California, Berkeley). EDTA, deuterium oxide (2H20), deuterium chloride (2HC1) and sodium deuteroxide (Na02H) were purchased from Aldrich Chemical Company.Fur sarnpies were dissolved in 500 pl 2H20. Sample 1 contained 16.4 mg and sample 2 17.5 mg Fur protein. Sample 1 was adjusted to pH 5.22 with 2HC1 and sample 2 to pH 7.40 with NaO'H. Subsequent pH changes were made by additions of NaO'H. The pH measurements were performed with a glass electrode; the quoted pH values were the actual readings measured to an accuracy of f0.005. The pH of Correspondence to R. J. P. Williams, Inorganic Chemistry Labordtory, University of Oxford, South Parks Road, Oxford OX1 3QR, UK Abbreviations. Fur, iron-uptake regulation protein; 1D and 2D, one-and two-dimensional. the solution was measured before and after recording each spectrum.'H-NMR experiments were carried out in Fourier-transform mode with suppression of the solvent peak and at a temperature of 300 K. A Bruker AM500 spectrometer was used for experiment 1 (sample 1) and a Bruker AM600 spectrometer for experiment 2 (sample 2); 800 scans for experiment 1 and 1000 scans for experiment 2 were collected in an 8 K memory. Dioxan was added as internal reference. pH titrations were performed in the range 5.22 -11.32 (experiment 1) and 7.40 -11.91 (experiment 2). Two-dimensional (2D) NMR meas...

The role of fur in the acid tolerance response of Salmonella typhimurium is physiologically and genetically separable from its role in iron acquisition

“…It is interesting that nuclear magnetic resonance studies examining ionization of the histidine residues suggest that Fur is a monitor of iron concentration and pH (40). Our results support this prediction.…”

“…Several laboratories have begun to define the functional regions of Fur involved in Fe(II) sensing and in binding DNA as a repressor (4,6,(40)(41)(42)50). The results of fusion protein studies combining different domains of Fur and the repressor cI857 indicate that the N terminus of Fur is involved in DNA binding while the C terminus is required for dimerization (50).…”

Isolation and analysis of a fur mutant of Neisseria gonorrhoeae

“…It is possible that this mutation affects the metal binding domain's ability to bind metals or its specificity in binding those metals. Alternatively, the mutation might affect the ability of this domain to transmit information regarding metal binding to the more amino-terminal proposed DNA binding domain (23,(54)(55)(56). This mutation also falls on the boundary of one of the alpha helix regions proposed by Wertheimer et al (75) and could alter this region's conformation as well.…”

“…Most biochemical analyses have utilized Fur purified from E. coli (72). Alpha helices near the amino terminus of the protein have been implicated in DNA binding (55,56), while a series of histidine residues near the middle and carboxyl terminus are thought to be involved in binding ferrous iron (54,56). Many genes repressed by Fur are involved in iron uptake, but iron starvation is also used by many pathogens as a signal that they are in a host environment with resultant expression of certain virulence factors.…”

Heme binds to and inhibits the DNA‐binding activity of the global regulator FurA from Anabaena sp. PCC 7120

“…Considering that histidine residues are common heme ligands that have been implicated in iron binding by this protein [12], and this iron binding is affected by the pH [25], we performed our studies at two different pHs (8 and 5). Upon addition of purified recombinant FurA to a solution of heme at pH 8, appearance of a peak at 416 nm in the difference spectrum was indicative of a complex formation between heme and FurA, since maximum absorbance of free heme under the same conditions is 384 nm (Fig.…”