Haemophore-mediated bacterial haem transport:: evidence for a common or overlapping site for haem-free and haem-loaded haemophore on its specific outer membrane receptor

Bacterial extracellular haemophores also named HasA for haem acquisition system form an independent family of haemoproteins that take up haem from host haeme carriers and shuttle it to specific receptors (HasR). Haemophore receptors are required for the haemophore-dependent haem acquisition pathway and alone allow free or haemoglobin-bound haem uptake, but the synergy between the haemophore and its receptor greatly facilitates this uptake. The three-dimensional structure of the Serratia marcescens holo-haemophore (HasA(SM)) has been determined previously and revealed that the haem iron atom is ligated by tyrosine 75 and histidine 32. The phenolate of tyrosine 75 is also tightly hydrogen bonded to the N delta atom of histidine 83. Alanine mutagenesis of these three HasA(SM) residues was performed, and haem-binding constants of the wild-type protein, the three single mutant proteins, the three double mutant proteins and the triple mutant protein were compared by absorption spectrometry to probe the roles of H32, Y75 and H83 in haem binding. We show that one axial iron ligand is sufficient to ligate haem efficiently and that H83 may become an alternative iron ligand in the absence of Y75 or both H32 and Y75. All the single mutant proteins retained the ability to stimulate haemophore-dependent haem uptake in vivo. Thus, the residues H32, Y75 and H83 are not individually necessary for haem delivery to the receptor. The binding of haem-free and haem-loaded HasA(SM) proteins to HasR(SM)-producing strains was studied. Both proteins bind to HasR(SM) with similar apparent K-d. The double mutant H32A-Y75A competitively inhibits binding to the receptor of both holo-HasA(SM) and apo-HasA(SM), showing that there is a unique or overlapping site on HasR(SM) for the apo- and holo-haemophores. Thus, we propose a new mechanism for haem uptake, in which haem is exchanged between haem-loaded haemophores; and unloaded haemophores bound to the receptor without swapping of haemophores; on the receptor.