Tight metal binding by humic acids and its role in biomineralization †

Analytical and thermodynamic data, EPR, FTIR, solution 1H and solid-state 13C cross polarization magic angle spinning NMR and solid-state extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge structure (XANES) spectra have been recorded for purified humic acids (HAs) isolated from a German peat (GHA), an Irish peat (IHA), an unpolluted New Hampshire bog soil (NHA) and their tightly bound copper(II), iron(III) and manganese(II) forms. Brief water washing of partly or fully metal-loaded HAs leaves 'tightly' bound metal in the isolated freeze-dried solids. Most of this metal is removed by washing with 0.1 M HCl, indicating acidic HA functional groups as principal metal binding sites. The number of nearest-neighbour atoms co-ordinated to tightly bound CuII (four), FeIII (six, probably with distorted geometry) and MnII (six, undistorted) in solid GHA, IHA and NHA were determined by XANES and EXAFS spectroscopy with reference standards. Isotherms measured at 20.0 °C and pH 2.4–3.2 with [M]total = 0.18–25.8 mM for tight, reversible Cu2+(aq), Fe3+(aq), and Mn2+(aq) binding by solid IHA and NHA fit the Langmuir model and give the pH-independent stoichiometric site capacities νi and equilibrium constants Ki for metal binding at specific HA sites i = A, B and C. Tight binding sites A, B and C of IHA are occupied by CuII, sites A and B by FeIII and site A by MnII, while only identical metal binding site A in NHA is tight enough to resist metal removal by brief water washing. A new helical HA molecular model based on the empirical formula C36H30N2O15·xH2O visualizes metal binding and the likely roles of HAs in biomineralization. Site A is suggested to be carboxylate, mixed ligands probably constitute site B, and site C is tentatively assigned as the interior of the HA helix. Binding free energies and EPR evidence suggest that Cu2+(aq), Fe3+(aq) and Mn2+(aq) rapidly transfer between specific HA binding sites. This affects rates of metal release and transfer to minerals.