Bacterial Iron Transport:  Coordination Properties of Azotobactin, the Highly Fluorescent Siderophore of Azotobacter v inelandii

• Published in: Inorganic chemistry 2004-02, Vol.43 (3), p.1137-1152
• Main Authors: Palanché, Tania, Blanc, Sylvie, Hennard, Christophe, Abdallah, Mohamed A, Albrecht-Gary, Anne-Marie
• Format: Article
• Language: English
• ISSN: 0020-1669; 1520-510X
• DOI: 10.1021/ic034862n

Azotobacter vinelandii, a nitrogen-fixing soil bacterium, secretes in iron deficiency azotobactin δ, a highly fluorescent pyoverdin-like chromopeptidic hexadentate siderophore. The chromophore, derived from 2,3-diamino-6,7 dihydroxyquinoline, is bound to a peptide chain of 10 amino acids:  (l)-Asp-(d)-Ser-(l)-Hse-Gly-(d)-β-threo-HOAsp-(l)-Ser-(d)-Cit-(l)-Hse-(l)-Hse lactone-(d)-Nδ-Acetyl, Nδ-HOOrn. Azotobactin δ has three different iron(III) binding sites which are one hydroxamate group at the C-terminal end of the peptidic chain (Nδ-Acetyl, Nδ-HOOrn), one α-hydroxycarboxylic function in the middle of the chain (β-threo-hydroxyaspartic acid), and one catechol group on the chromophore. The coordination properties of its iron(III) and iron(II) complexes were measured by spectrophotometry, potentiometry, and voltammetry after the determination of the acid−base functions of the uncomplexed free siderophore. Strongly negatively charged ferric species were observed at neutral p[H]'s corresponding to a predominant absolute configuration Λ of the ferric complex in solution as deduced from CD measurements. The presence of an α-hydroxycarboxylic chelating group does not decrease the stability of the iron(III) complex when compared to the main trishydroxamate siderophores or to pyoverdins. The value of the redox potential of ferric azotobactin is highly consistent with a reductive step by physiological reductants for the iron release. Formation and dissociation kinetics of the azotobactin δ ferric complex point out that both ends of this long siderophore chain get coordinated to Fe(III) before the middle. The most striking result provided by fluorescence measurements is the lasting quenching of the fluorophore in the course of the protonation of the ferric azotobactin δ complex. Despite the release of the hydroxyacid and of the catechol, the fluorescence remains indeed quenched, when iron(III) is bound only to the hydroxamic acid, suggesting a folded conformation at this stage, around the metal ion, in contrast to the unfolded species observed for other siderophores such as ferrioxamine or pyoverdin PaA.