Deciphering the Role of Glycine Betaine in Modulating the Kinetics and Thermodynamics of Iron-Binding and Release by Human Serum Transferrin at Physiological, Endosomal, and Acidic pH Levels

Human serum transferrin (hSTf) reversibly binds to Fe3+ ions and delivers them to cells in the presence of physiological anions (chloride, ATP, citrate). In humans, glycine betaine (GB) serves as a metabolic methyl donor in the liver. As the liver is the primary site of hSTf synthesis, GB’s influence on hSTf is crucial for maintaining the protein’s structural integrity and regulating iron metabolism. This study deciphers how GB affects the kinetics and thermodynamics of iron binding and release by hSTf, both in the absence and in the presence of chloride anions, at physiological pH 7.4, endosomal pH 5.5, and acidic pH levels below 5.0. Analysis of the effects of GB on equilibrium and kinetic parameters for iron release of hSTf and molecular dynamics (MD) simulations of hSTf with GB yielded significant results: (i) at pH 7.4 and 5.5, the experimental and MD simulation studies suggest that the preferential exclusion of GB from the protein enhances the stability of the Fe3+-hSTf complex in diferric-hSTf (Fe2hSTf); (ii) at pH 7.4 and 5.5, the enthalpic stabilization effects of GB retard the rate of iron release from monoferric-N-lobe of Fe2hSTf (FeNhSTf); (iii) at acidic pH (<5), the preferential binding of GB to Fe2hSTf reduces the stability of the Fe3+-hSTf complex and accelerates the rate of iron release from Fe2hSTf; (iv) GB reduces the conformational flexibility and backbone fluctuation to stabilize Fe2hSTf conformation at pH 7.4 and 5.5, while it induces conformational changes that lead to higher protein fluctuations and flexibility at pH 4.5; (v) under acidic pH conditions (<5), nonsynergistic chloride anion reduces the stability of the Fe3+-hSTf complex, and the addition of GB exhibits an additive impact on chloride effect to reduce the stability of the Fe3+-hSTf complex in Fe2hSTf. Thermodynamic analysis of isothermal titration calorimetry (ITC) profiles revealed that GB enhances the binding affinity of Fe3+ to apo-hSTf at physiological pH 7.4. Additionally, the investigation of GB’s effects on the average fluorescence lifetime of Fe2hSTf (τavg) at pH 7.4 and pH 5.5 reveals that GB forms ground-state interactions with Fe2hSTf at physiological pH 7.4 while engaging in excited-state interactions at endosomal pH 5.5.