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This work focuses on the relationship between the coordination chemistry and antimicrobial activity of Zn(II) and Cu(II) complexes of histatin 5 and the products of its hydrolysis: its N-terminal fragment (histatin 5-8) and C-terminal fragment (histatin 8). Cu(II) coordinates in an albumin-like binding mode and Zn(II) binds to up to 3 His imidazoles. The antimicrobial activity of histatins and their metal complexes (i) strongly depends on pH - they are more active at pH 5.4 than at 7.4; (ii) the complexes and ligands alone are more effective in eradicating Gram-positive bacteria than the Gram-negative ones, and (iii) Zn(II) coordination is able to change the structure of the N-terminal region of histatin 5 (histatin 5-8) and moderately increase all of the studied histatins' antimicrobial potency.

The histidine-rich salivary peptides of the histatin family are known to bind copper (Cu) and other metal ions in vitro; however, the details of these interactions are poorly understood, and their implications for in vivo antifungal activity have not been established. Here, we show that the availability of Cu during exposure of Candida albicans to histatin-5 (Hist-5) modulates its antifungal activity. Antifungal susceptibility testing revealed that co-treatment of Hist-5 with Cu improved the EC 50 from ∼5 to ∼1 μM, whereas co-treatment with a high-affinity Cu-specific chelator abrogated antifungal activity. Spectrophotometric titrations revealed two previously unrecognized Cu(I)-binding sites with apparent K d values at pH 7.4, ∼20 nM, and confirmed a high-affinity Cu(II)-binding site at the Hist-5 N-terminus with an apparent K d of ∼8 pM. Evaluation of a series of His-to-Ala full-length and truncated Hist-5 peptides identified adjacent His residues (bis-His) as critical anchors for Cu(I) binding, with the presence of a third ligand revealed by X-ray absorption spectroscopy. On their own, the truncated peptides were ineffective at inhibiting the growth of C. albicans, but treatment with supplemental Cu resulted in EC 50 values down to ∼5 μM, approaching that of full-length Hist-5. The efficacy of the peptides depended on an intact bis-His site and correlated with Cu(I) affinity. Together, these results establish new structure-function relationships linking specific histidine residues with Cu binding affinity and antifungal activity and provide further evidence of the involvement of metals in modulating the biological activity of these antifungal peptides.

Natural antimicrobial peptides represent a primordial mechanism of immunity in both vertebrate and nonvertebrate organisms. Among them, histatins belong to a family of human salivary metal-binding peptides displaying potent antibacterial, antifungal and wound-healing activities. These properties, along with the ability of histatins to inhibit collagenases and cysteine proteases, have attracted much attention for their potential use in the treatment of several oral diseases. This review critically assesses the studies carried out to date in order to provide a comprehensive and systematic vision of the information accumulated so far. In particular, the relationship between metal-binding and peptide activity is extensively analysed. The review provides important clues for developing possible therapeutic applications of histatins and their synthetic peptide analogues by creating a set of necessary resource materials to support investigators and industries interested in exploiting their unique properties., (© 2013 FEBS.)

Histatin-5 (Hn5) is an antimicrobial salivary peptide of 24 amino acids. Two specific metal-binding sites were revealed with electronic, NMR, and EPR spectroscopy. The complex Cu(2)(II)-Hn5 effectively oxidizes catechol, exhibiting enzyme-like kinetics (k(cat)=0.011 and 0.060 s(-1) and k(cat)/K(m)=19 and 50 M(-1)s(-1) without and with 12.8mM H(2)O(2), respectively). The significant oxidative activity may contribute to the biological activity of this antibiotic metallopeptide.

The ability of the histidine-rich peptides, histatin-5 (Hst-5) and histatin-8 (Hst-8), to support the generation of reactive oxygen species during the Cu-catalyzed oxidation of ascorbate and cysteine has been evaluated. High levels of hydrogen peroxide (70-580 mol/mol Cu/h) are produced by aqueous solutions containing Cu(II), Hst-8 or Hst-5, and a reductant, either ascorbate or cysteine, as determined by the postreaction Amplex Red assay. When the reactions are conducted in the presence of superoxide dismutase, the total hydrogen peroxide produced is decreased, more so in the presence of the peptides (up to 50%), suggesting the intermediacy of superoxide in these reactions. On the other hand, the presence of sodium azide or sodium formate, traps for hydroxyl radicals, has no appreciable effect on the total hydrogen peroxide production for the Cu-Hst systems. EPR spin-trapping studies using 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO) in the cysteine-Cu(II) reactions reveal the formation of the CYPMPO-hydroperoxyl and CYPMPO-hydroxyl radical adducts in the presence of Hst-8, whereas only the latter was observed with Cu alone.