A histidine-rich metal binding domain at the N terminus of Cu,Zn-superoxide dismutases from pathogenic bacteria

A group of Cu,Zn-superoxide dismutases from pathogenic bacteria is characte rized by histidine-rich N-terminal extensions that are in a highly exposed and mobile conformation. This feature allows these proteins to be readily p urified in a single step by immobilized metal affinity chromatography. The Cu,Zn-superoxide dismutases from both Haemophilus ducreyi and Haemophilus p arainfluenzae display anomalous absorption spectra in the visible region du e to copper binding at the N-terminal region. Reconstitution experiments of copper-free enzymes demonstrate that, under conditions of limited copper a vailability, this metal ion is initially bound at the N-terminal region and subsequently transferred to an active site. Evidence is provided for inter molecular pathways of copper transfer from the N-terminal domain of an enzy me subunit to an active site located on a distinct dimeric molecule. Incuba tion with EDTA rapidly removes copper bound at the N terminus but is much l ess effective on the copper ion bound at the active site. This indicates th at metal binding by the N-terminal histidines is kinetically favored, but t he catalytic site binds copper with higher affinity. We suggest that the hi stidine-rich N-terminal region constitutes a metal binding domain involved in metal uptake under conditions of metal starvation in vivo. Particular bi ological importance for this domain is inferred by the observation that its presence enhances the protection offered by periplasmic Cu,Zn-superoxide d ismutase toward phagocytic killing.