Stability constants of pseudobactin complexes with transition metals

The stability constants of the fluorescent siderophore pseudobactin St3 (PSB₃) produced by Pseudomonas putida strain 3 with Fe³, Cu²⁺, Mn²⁺, Zn²⁺, and Fe²⁺ were determined using fluorscence spectroscopy and potentiometric titrations. Stability constants for PSB₃, with Fe³⁺, Fe²⁺, Mn²⁺, and Zn²⁺ were determined based on titration to saturation of a PSB₃ ligand solution with either of the metals, then back-titrating the solution with an EDTA solution along with simultaneous measurements of fluorescene and visible spectra. A nonlinear curve fitting between the fluorescence intensity (Y) and the concentration (C) of the metal or EDTA was used for the determination of the concentrations of the free and complexed ligands. The overall stability constant (log K_ML) obtained for Fe³⁺ by this method was the same as that obtained by visible spectroscopy. The log K_ML values obtained by fluorescence spectroscopy for Mn²⁺ and Zn³⁺ (17.5 and 19.4, respectively) were in good agreement with log K_ML values obtained by potentiometric titration (17.3 and 19.8, respectively). It was found that two chromatography bands obtained from PSB₃ solutions isolated and purified from cultures grown under different conditions chelated Fe³⁺ with practically the same affinity. The log K_ML of PSB₃ with Fe³⁺, Zn²⁺, Cu²⁺, Mn²⁺, and Fe²⁺ were calculated using the protonation constants determined by potentiometry (pK_e1 = 11.28, pk_e2 = 7.74, pK_e3 = 5.35) and were found to be 29.6, 19.8, 22.3, 17.3, and 8.3, respectively.