Heme binding by a bacterial repressor protein, the gene product of the ferric uptake regulation (fur) gene of Escherichia coli.
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.
Smith
A
A
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City 64110, USA. annsmith@cctr.umkc.edu
Hooper
N I
NI
Shipulina
N
N
Morgan
W T
WT
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D006418:Heme / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism; D008665:Metalloporphyrins / Q000378:metabolism; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
D001426:Bacterial Proteins; D008665:Metalloporphyrins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D006418:Heme; D008345:Manganese; D015032:Zinc