Most in vitro iron mobilization studies from ferritin have been performed in aqueous buffered solutions using a variety of reducing substances. The kinetics of iron mobilization from ferritin in a medium that resembles the complex milieu of cells could dramatically differ from those in aqueous solutions, and to our knowledge, no such studies have been performed. Here, we have studied the kinetics of iron release from ferritin in fresh yeast cell lysates and examined the effect of cellular metabolites on this process. Our results show that iron release from ferritin in buffer is extremely slow compared to cell lysate under identical experimental conditions, suggesting that certain cellular metabolites present in yeast cell lysate facilitate the reductive release of ferric iron from the ferritin core. Using filtration membranes with different molecular weight cut-offs (3, 10, 30, 50, and 100 kDa), we demonstrate that a cellular component >50 kDa is implicated in the reductive release of iron. When the cell lysate was washed three times with buffer, or when NADPH was omitted from the solution, a dramatic decrease in iron mobilization rates was observed. The addition of physiological concentrations of free flavins, such as FMN, FAD, and riboflavin showed about a two-fold increase in the amount of released iron. Notably, all iron release kinetics occurred while the solution oxygen level was still high. Altogether, our results indicate that in addition to ferritin proteolysis, there exists an auxiliary iron reductive mechanism that involves long-range electron transfer reactions facilitated by the ferritin shell. The physiological implications of such iron reductive mechanisms are discussed.
Keywords: FAD; FMN; NADPH; ferritin; flavoenzymes; iron mobilization; kinetics; labile iron pool; riboflavin; yeast cell lysate.