Gram-negative bacteria use specific heme uptake systems, relying on outer membrane receptors and excreted heme-binding proteins (hemophores) to scavenge and actively transport heme. To unravel the unknown molecular details involved, we present 3 structures of the Serratia marcescens receptor HasR in complex with its hemophore HasA. The transfer of heme over a distance of 9 Å from its high-affinity site in HasA into a site of lower affinity in HasR is coupled with the exergonic complex formation of the 2 proteins. Upon docking to the receptor, 1 of the 2 axial heme coordinations of the hemophore is initially broken, but the position and orientation of the heme is preserved. Subsequently, steric displacement of heme by a receptor residue ruptures the other axial coordination, leading to heme transfer into the receptor.heme binding ͉ iron uptake ͉ membrane protein ͉ membrane transport ͉ protein complex
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.
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