Synthetic, potentiometric and spectroscopic studies of chelation between Fe(III) and 2,5-DHBA supports salicylate-mode of siderophore binding interactions

Abstract

Catecholate type enterobactin, a prototype siderophore, comprises 2,3-dihydroxybenzoic acid (2,3-DHBA) cyclically linked to serine in E. coli. The existence of iron-chelating ligands in humans is a recent discovery, however, the basic chemical interactions between 2,5-dihydroxybenzoic acid and Fe(III) ion remain poorly understood. Achieving an accurate description of the fundamental Fe(III) binding properties of 2,5-DHBA is essential for understanding its role in iron transport mechanisms. Here, we show that 2,5-DHBA binds iron in a salicylate mode via a two-step kinetic mechanism by UV spectroscopy. Complexation between Fe(III) salt and 2,5-DHBA initially occurs at 1:1 ratio (of ligand to metal) and binding resulting in higher-order complexes continues at higher concentrations. Through potentiometric measurements we quantify the distribution of Fe(III)-2,5-DHBA complexes with 1:1, 1:2 and 1:3 stoichiometry. The formation of 1:3 complexes is further supported through high-resolution mass spectrometry. Further, using kinetic and equilibrium UV spectroscopy, we report Fe(III)-2,5-DHBA complex formation at a pH range of 2.5-9.0 at 298.15K in water. Maximum complexation occurred at a pH range of 4.5-6.5 consistent with deprotonation of the carboxylic acid proton. Equilibrium measurements and stopped-flow kinetics show that complexation rate constants were independent of concentrations of 2,5-DHBA. Together the data supports a model in which the rate-determining step involves rearrangement of ligands on an initial complex formed by reversible binding between the carboxylate group of 2,5-DHBA and Fe(III).

Keywords: 2,5-DHBA; Gentisic acid; Iron chelation; Salicylate mode; UV–Vis spectroscopy.