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

Abstract

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 Cu^II (four), Fe^III (six, probably with distorted geometry) and Mn^II (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 ν_i and equilibrium constants K_i for metal binding at specific HA sites i = A, B and C. Tight binding sites A, B and C of IHA are occupied by Cu^II, sites A and B by Fe^III and site A by Mn^II, 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.