A Histidine-rich Metal Binding Domain at the N Terminus of Cu,Zn-Superoxide Dismutases from Pathogenic Bacteria

Abstract

A group of Cu,Zn-superoxide dismutases from pathogenic bacteria is characterized by histidine-rich N-terminal extensions that are in a highly exposed and mobile conformation. This feature allows these proteins to be readily purified in a single step by immobilized metal affinity chromatography. The Cu,Zn-superoxide dismutases from both Haemophilus ducreyi and Haemophilus parainfluenzae display anomalous absorption spectra in the visible region due to copper binding at the N-terminal region. Reconstitution experiments of copper-free enzymes demonstrate that, under conditions of limited copper availability, this metal ion is initially bound at the N-terminal region and subsequently transferred to an active site. Evidence is provided for intermolecular pathways of copper transfer from the N-terminal domain of an enzyme subunit to an active site located on a distinct dimeric molecule. Incubation with EDTA rapidly removes copper bound at the N terminus but is much less effective on the copper ion bound at the active site. This indicates that metal binding by the Nterminal histidines is kinetically favored, but the catalytic site binds copper with higher affinity. We suggest that the histidine-rich N-terminal region constitutes a metal binding domain involved in metal uptake under conditions of metal starvation in vivo. Particular biological importance for this domain is inferred by the observation that its presence enhances the protection offered by periplasmic Cu,Zn-superoxide dismutase toward phagocytic killing.

On the possible roles of N-terminal His-rich domains of Cu,Zn SODs of some Gram-negative bacteria

“…The increasing number of structurally characterized prokaryotic Cu,Zn superoxide dismutases show a considerable structural variability between proteins from different bacteria, therefore individual enzyme variants may exhibit unique properties [3][4][5][6][7][8]. Such species-specific differences include alteration in the quaternary structure [3,4], mutations in the active site ligands [5][6][7], deletion or insertion in the major enzyme loops [6,7], or the introduction of additional domains at the N-or C-terminus of the enzyme [8]. The Cu,Zn SODs from a few Gram-negative bacteria possess histidine-rich N-terminal extensions, which show typical features of high affinity divalent metal binding domains [8].…”

“…Such species-specific differences include alteration in the quaternary structure [3,4], mutations in the active site ligands [5][6][7], deletion or insertion in the major enzyme loops [6,7], or the introduction of additional domains at the N-or C-terminus of the enzyme [8]. The Cu,Zn SODs from a few Gram-negative bacteria possess histidine-rich N-terminal extensions, which show typical features of high affinity divalent metal binding domains [8].…”

“…The Cu,Zn SODs of certain bacteria (e.g. H. parainfluenzae, H. influenzae, N. meningitidis) possess an N-terminal HxH sequence [8], a well-known high affinity copper(II) binding site (ATCUN motif) found in the N-terminus of several naturally occurring copper-binding proteins, such as human serum albumin [10]. Further interesting examples of such metal binding regions are present in the enzymes isolated from Haemophilus ducreyi and Actinobacillus pleuropneumoniae.…”

“…The N-terminal domain of H. ducreyi Cu,Zn SOD is constituted by the juxtaposition of a histidine-and a methionine-rich sequence (HGDHMHNHDTKMDTMSKDMMSME….) [7,8]. Both subdomains are able to bind copper, but the histidine-rich region has a marked preference for Cu(II) binding, while the methionine-rich region preferentially binds Cu(I) [11].…”

“…Although, the first six amino acids has analogous sequence (HxDHxH), the Cu,Zn SOD from Actinobacillus pleuropneumoniae has different N-terminal extension (HADHDHKKADNSSVE…, [8]), the methionine-rich sequence is completely absent and contains only three histidine units (instead of four in H. ducreyi Cu,Zn SOD). If the above mentioned chaperoning role of the N-terminal His-rich region of Gram-negative bacteria can be generalized, the N-terminal fragment of Cu,Zn SOD from A. pleuropneumoniae should have comparable metal binding ability with the corresponding H. ducreyi peptide, in spite of the reduced number of the available donor sites.…”