Lactoferrin as an active coordination scaffold for ruthenium(iii)

Abstract

This study sought to elucidate the molecular principles by which bovine lactoferrin (bLF) governs ruthenium(III) coordination and to assess how protein-guided binding translates into structural and biological effects. The protein was modified using potassium aquapentachlororuthenate(III) (K₂[RuCl₅(H₂O)]) as a metal precursor. The obtained LF–Ru systems were comprehensively examined using spectroscopic (spectrofluorimetry, DLS, ATR-FTIR, SRCD), spectrometric (ICP-OES), microscopic (LM, SEM-EDS, TEM-EDS), scattering (SAXS), and electrophoretic (SDS-PAGE) techniques. Antibacterial activity was evaluated for LF and LF–100Ru against Gram-positive (Enterococcus faecalis) and Gram-negative (Escherichia coli, Klebsiella pneumoniae) bacteria. The cellular response to LF–Ru complexes was investigated using murine fibroblast (L929), human hepatocellular carcinoma (HepG2), and human colorectal adenocarcinoma (Caco-2) cell lines. Molecular docking indicated favorable Ru(III) binding in histidine-containing regions related to native metal-binding motifs. Molecular dynamics simulations supported conformational stability upon metal binding, whereas quantum-mechanical calculations on simplified residue models were used only to assess qualitative donor preferences. The adsorption isotherm was better described by the Langmuir model, indicating saturation-like binding of Ru(III) to accessible LF regions at low and moderate loading. At the highest precursor concentrations, BET-style analysis together with TEM observations suggested additional secondary Ru accumulation within the protein matrix. Fluorescence quenching demonstrated strong interactions in the LF–Ru system (K_a = 1.789 × 10⁴ M⁻¹). Desorption studies indicated only minor metal release from LF–100Ru after incubation in simulated gastric and intestinal fluids. Importantly, LF–100Ru reduced the viability of HepG2 and Caco-2 cells while showing comparatively low toxicity toward normal L929 fibroblasts under the tested conditions, indicating a differential cellular response.