Purification and characterization of an extracellular heme-binding protein, HasA, involved in heme iron acquisition.
Many bacterial hemoproteins involved in heme acquisition have been isolated recently, comprising outer membrane receptors and extracellular heme-binding protein. The mechanisms by which these proteins extract heme have not been described up to now. One such protein, HasA, which can bind free heme as well as capture it from hemoglobin, is secreted by the Gram-negative bacteria Serratia marcescens under iron deficiency conditions. The fact that HasA does not present sequence similarities with other known hemoproteins suggests that it possesses a new type of heme binding site. This work describes the main physicochemical properties of HasA, essential for understanding its function. HasA is a monomer of 19 kDa that binds one b heme per molecule with high affinity. The electron paramagnetic resonance spectra indicate that the heme iron is in a low-spin ferric state and that the two iron axial ligands are His and His-. The low oxidation-reduction potential value (-550 mV vs standard hydrogen electrode) of the heme bound to HasA suggests that heme could be exposed to the solvent. According to circular dichroism data, the binding of heme does not seem to modify the conformation of HasA.
Izadi
N
N
Laboratoire de Résonance Magnétique Nucléaire et Unité de BiochimieCellulaire, CNRS URA 1129, Institut Pasteur, Paris, France. nizadi@pasteur.fr
Henry
Y
Y
Haladjian
J
J
Goldberg
M E
ME
Wandersman
C
C
Delepierre
M
M
Lecroisey
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002352:Carrier Proteins; D002417:Cattle; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D002352:Carrier Proteins; C093956:HasA protein, Serratia marcescens; D008565:Membrane Proteins; D006418:Heme; D007501:Iron