A novel heme protein, the Cu,Zn-superoxide dismutase from Haemophilus ducreyi

Haemophilus ducreyi, the causative agent of the genital ulcerative disease known as chancroid, is unable to synthesize heme, which it acquires from hu mans, its only known host. Here we provide evidence that the periplasmic Cu ,Zn-superoxide dismutase from this organism is a heme-binding protein, unli ke all the other known Cu,Zn-superoxide dismutases from bacterial and eukar yotic species. When the H. ducreyi enzyme was expressed in Escherichia coli cells, grown in standard LB medium, it contained only limited amounts of h eme covalently bound to the polypeptide but was able efficiently to bind ex ogenously added hemin. Resonance Raman and electronic spectra at neutral pH indicate that H. ducreyi Cu,Zn-superoxide dismutase contains a 6-coordinat ed low spin heme, with two histidines as the most likely axial ligands. By site-directed mutagenesis and analysis of a structural model of the enzyme, we identified as a putative axial ligand a histidine residue (His-64) that is present only in the H. ducreyi enzyme and that was located at the botto m of the dimer interface. The introduction of a histidine residue in the co rresponding position of the Cu,Zn-superoxide dismutase from Haemophilus par ainfluenzae was not sufficient to confer the ability to bind heme, indicati ng that other residues neighboring His-64 are involved in the formation of the heme-binding pocket. Our results suggest that periplasmic Cu,Zn-superox ide dismutase plays a role in heme metabolism of H. ducreyi and provide fur ther evidence for the structural flexibility of bacterial enzymes of this c lass.