Heme binding by a bacterial repressor protein, the gene product of the ferric uptake regulation (fur) gene ofEscherichia coli

Abstract

The fur gene product, Fur, of Escherichia coli is a repressor when it binds Fe(II). Since heme and iron metabolism are closely linked and Fur is rich in histidine, a ligand for heme, the binding of heme to Fur was investigated. The oxidized Fur-heme complex is stable and low spin with a Soret maximum at 404 nm and no 620-nm band. CO coordinates with the reduced heme-Fur complex, causing a shift from 412 nm to 410 nm, and stabilizes it, increasing the half-life from 5 to 15 min. Circular dichroism (CD) spectra in the Soret region show heme bound in an asymmetric environment in Fur, both in the oxidized and reduced-CO forms. Quenching of tyrosine fluorescence by heme revealed rapid, tight binding (Kd < 1 microM) with an unusual stoichiometry of 1 heme:1 Fur dimer. Fur binds Mn(II), a model ligand for the endogenous Fe(II), much more weakly (Kd > 80 microM). Far-ultraviolet CD spectroscopy showed that the alpha-helix content of apo-Fur decreases slightly with heme binding, but increases with Mn(II) binding. Competition experiments indicated that heme interacts with Fur dimers at the same site as Mn(II) and can displace the metal. In contrast to Mn(II), Zn(II) did not quench the tyrosine fluoroescence of Fur, affected the CD spectrum less than Mn(II), but did bind in a manner which prevented heme from binding. In sum, Fur not only binds heme and Zn(II) with sufficient affinity to be biologically relevant, but the interactions that occur between these ligands and their effects on Mn(II) binding need to be taken into account when addressing the biological function of Fur.

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

“…4C, lanes 4 and 8). This agrees only partially with previous observations of Smith et al, suggesting that heme most likely binds at or near the same site as Mn 2þ rather than to an independent site, since binding of heme to Fur from E. coli prevents Mn 2þ binding in vitro [12]. From our results, we cannot infer that both species compete for the same site although it seems that the binding of one of them affects the affinity of the complex for the other.…”

“…In fact, the K d values measured in E. coli for the Mn 2þ Fur complex (85 lM) [40] and the heme-Fur one (<l lM) [12] indicate that a smaller concentration of heme than Mn 2þ is sufficient to induce the complex formation.…”

“…In particular, histidine has shown to be essential in the reversible heme-protein binding that takes place in HasA protein from Serratia marcescens [10], a hemophore that binds free or hemoprotein bound heme with high affinity and delivers it to a specific outer membrane receptor HasR [11]. It has been suggested that histidine residues might be involved as well in the binding of heme by Fur (ferric uptake regulator) from Escherichia coli [12]. Fur is a DNA-binding protein that regulates iron responsive genes [13].…”

“…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.…”

Genetics and regulation of two distinct haem-uptake systems, phu and has, in Pseudomonas aeruginosa The GenBank accession numbers for the sequences reported in this paper are AF055999, AF127222, and AF127223.

“…Since haem binding of Fur has been observed in vitro for E. coli Fur (Smith et al, 1996), a model was tested in which the transcripts T1 and T2 would be repressed by haem. However, the addition of haem to otherwise irondepleted medium did not result in a selective repression of T1\T2, but had a non-discriminative negative effect on T1\T2 as well as on phuS expression.…”