Structural and Biochemical Investigations of the Mechanism of Heme Capture by the Hemophore HasAp from Pseudomonas aeruginosa

Abstract

HasAp is a hemophore secreted by Pseudomonas aeruginosa to the extracellular media under iron limited conditions to sequester heme from the host cell. The heme HasAp complex is captured by the specific cell surface receptor HasR for subsequent internalization. X-ray and solution NMR structures of holo and apo hemophores have been solved but so far no studies have been done to elucidate the mechanism of heme loading. In the apo form, the loop bearing the distal heme iron ligand His 32 is located nearly 30 Å away from its position in the holo form. In contrast, the loop bearing the proximal ligand Tyr 75 maintains structural integrity in the apo and holo forms. This study aimed at investigating the sequential steps that lead to heme binding by HasAp and the role played by the axial-ligand bearing loops in the process of heme capture. A chromatographic method was developed to isolate and purify wild type and H32A apo HasAp. Heme binding was monitored by stopped flow spectroscopy. The results show that heme loading proceeds in two discrete kinetic phases; an initial rapid phase which entail loading of the heme onto the Tyr 75 loop followed by a slow phase where the His 32 loop undergoes a large conformational rearrangement and subsequent coordination of the heme. Molecular dynamic simulations provided more insights into the molecular motions that relay the presence of the heme in the Tyr 75 loop to the His 32 loop. To investigate the role played by the axial ligand bearing loops in heme binding, crystal structures and solution NMR studies of the apo and holo H32A-HasAp, as well as H32A-imidazole complex were carried out. The results reveal that the His 32 loop assumes a position similar to the wild type protein even in the absence of a coordinating residue. This implies that the presence of the heme in the Tyr 75 loop triggers the relocation of the His 32 loop and that this loop is important in protecting the macrocycle from the aqueous media. The crystal structures of Y75A and H83A show that the proteins assume the original  fold although there are some conformational changes in the His 32 loop and in the secondary structure elements in regions of the protein implicated to interact with the receptor during heme release. These observations imply that information is relayed between the Tyr 75 loop and the secondary structure elements affected likely via the His 32 loop. It was also established that His 83 does not necessarily coordinate the heme in the absence of Tyr 75. Heme transfer experiments using methemalbumin as a heme source revealed that the rate of heme release from methemalbumin correlates with the rate of heme uptake by apo-HasAp. These rates are comparatively higher than the rate of heme uptake from other host heme proteins; however, they are comparable to the rate of dissociation of heme from methemalbumin in the absence of the hemophore. These results mean that methemalbumin is likely the potential target for HasAp and that heme transfers by passive diffusion and is affinity driven.