PURIFICATION AND CHARACTERIZATION OF AN EXTRACELLULAR HEME-BINDING PROTEIN, HASA, INVOLVED IN HEME IRON ACQUISITION

Many bacterial hemoproteins involved in heme acquisition have been iso lated recently, comprising outer membrane receptors and extracellular heme-binding protein. The mechanisms by which these proteins extract h eme have not been described up to now. One such protein, HasA, which c an bind free heme as well as capture it from hemoglobin, is secreted b y 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 posseses a new type of heme binding site. This work describes the main physicochemical prope rties of HasA, essential for understanding its function. HasA is a mon omer of 19 kDa that binds one b heme per molecule with high affinity. The electron paramagnetic resonance spectra indicate that the heme iro n is in a low-spin ferric state and that the two iron axial ligands ar e His and His(-). The low oxidation-reduction potential value (-550 mV vs standard hydrogen electrode) of the heme bound to HasA suggests th at heme could be exposed to the solvent. According to circular dichroi sm data, the binding of heme does not seem to modify the conformation of HasA.