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BIOPHYSICAL CHARACTERIZATION OF IRON SIDEROPHORES AND IRON SIDEROPHORE MIMICS
- Abstract:
This work examines inorganic and bioinorganic aspects of siderophore mediated iron transport in microbial systems. Various stages of iron(III) acquisition, transport and release are studied through in vitro modeling in an effort to elucidate the mechanisms involved in biological iron mobility. Two synthetic siderophore mimics, including a saccharide-based desferrichrome and a heterotripodal (mono-hydroxamate, bis-catecholate) siderophore analogue are studied with respect to their aqueous solution behavior and iron(III) complex equilibria. These siderophore analogues are characterized in aqueous solution through determination of ligand pKa values and iron(III) binding constants using spectrophotometric and potentiometric titration techniques. In the case of the heterotripodal ligand, proton dependent iron(III)-ligand equilibrium constants were determined using a model based on the sequential protonation of the iron(III)-ligand complex. The results of these experiments were used to calculate the pH dependent speciation, the formation constant log and pM value with iron(III). The implications of these results, along with the ability of these synthetic siderophore mimics to deliver iron to living cells through growth promotion assays using various cell lines, are discussed.
The release of iron from ferri-siderophores is explored using a redox facilitated ligand exchange hypothesis. In these studies, the mechanism of iron release from ferrioxamine B, a well studied prototypical siderophore, is examined in vitro. Spectroscopic methods are used to explore the kinetics and thermodynamics of iron reduction and release, which is facilitated by the concurrent oxidation of glutathione, ascorbate or NADH. In the case of glutathione and ascorbate, a mechanism is described in which a ternary complex is formed between ferrioxamine B and an iron(II) chelator in a rapidly established pre-equilibrium step, which is followed by rate limiting reduction of the ternary complex by glutathione or ascorbate. Reduction is followed by a rapid ligand exchange step where iron is released from ferrioxamine B through ligand exchange with the iron(II) chelator.
Interestingly, while glutathione and ascorbate require the formation of a ternary complex to effectively reduce the iron(III) in ferrioxamine B, NADH is capable of reducing either the ternary complex or ferrioxamine B directly in the presence of an iron(II) chelator.
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