Haemophilus influenzae is an obligate human commensal/pathogen. This haem auxotroph must acquire haem from its host to sustain aerobic growth. Haem-haemopexin complexes are one of the potential sources of haem for this microorganism. Haemopexin is a glycoprotein that binds haem with high affinity (subpicomolar Kd) and involved in haem recycling. HxuA, a cell surface protein, is the key to haem acquisition from haemopexin. In this study, we reconstituted a functional Hxu system from H. influenzae in Escherichia coli K-12 that mediated active haem transport across the outer membrane from haem-haemopexin, in the presence of the inner membrane energy-transducing TonB-ExbB-ExbD complex from H. influenzae. A secreted variant of HxuA, HxuA(dm), was produced in E. coli. HxuA(dm) functionally complemented an hxuA mutant of H. influenzae for haem-haemopexin acquisition. HxuA(dm) interacted with haemopexin and haem-haemopexin, with which it formed high-affinity, stoichiometric complexes. Following the interaction between haem-haemopexin and HxuA(dm), haem was no longer bound to its initial high-affinity site and became accessible to its cognate haem receptor, HxuC. HxuA(dm) and the HxuA(dm)-haemopexin complex do not appear to bind haem at detectable levels (affinities below 10(6) M(-1)). HxuA thus appears to 'release' haem from haem-haemopexin complexes and to prevent haem sequestering by haemopexin.
© 2011 Blackwell Publishing Ltd.