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 Fe, Cu2+, Mn2+, Zn2+, and Fe2+ were determined using fluorescence 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 fluorescence 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 spectrosopy. The log K(ML) values obtained by fluorescence spectroscopy for Mn2+ and Zn2+ (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(a1) = 11.28, pK(a2) = 7.74, pK(a3) = 5.35) and were found to be 29.6, 19.8, 22.3, 17.3, and 8.3, respectively.