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 humans, its only known host. Here we provide evidence that the periplasmic Cu,Zn-superoxide dismutase from this organism is a heme-binding protein, unlike all the other known Cu,Zn-superoxide dismutases from bacterial and eukaryotic species. When the H. ducreyi enzyme was expressed in Escherichia coli cells grown in standard LB medium, it contained only limited amounts of heme covalently bound to the polypeptide but was able efficiently to bind exogenously added hemin. Resonance Raman and electronic spectra at neutral pH indicate that H. ducreyi Cu,Zn-superoxide dismutase contains a 6-coordinated 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 bottom of the dimer interface. The introduction of a histidine residue in the corresponding position of the Cu,Zn-superoxide dismutase from Haemophilus parainfluenzae was not sufficient to confer the ability to bind heme, indicating that other residues neighboring His-64 are involved in the formation of the heme-binding pocket. Our results suggest that periplasmic Cu,Zn-superoxide dismutase plays a role in heme metabolism of H. ducreyi and provide further evidence for the structural flexibility of bacterial enzymes of this class.
Pacello
F
F
Dipartimento di Biologia and INFM, Università di Roma Tor Vergata, Via della Ricerca Scientifica, 00133 Roma, Italy.
Langford
P R
PR
Kroll
J S
JS
Indiani
C
C
Smulevich
G
G
Desideri
A
A
Rotilio
G
G
Battistoni
A
A
D000595:Amino Acid Sequence; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006191:Haemophilus ducreyi / Q000201:enzymology*; D006418:Heme / Q000737:chemistry*; D006427:Hemin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012984:Software; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D008024:Ligands; D011994:Recombinant Proteins; D006418:Heme; D006427:Hemin; D013482:Superoxide Dismutase