Iron Mobilization from Ferritin in Yeast Cell Lysate and Physiological Implications

Abstract

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.

Figure 1

Iron mobilization kinetics from ferritin in yeast cell lysate (YCL). (A) Absorbance change of the (Fe²⁺–(ferrozine)₃) complex following the exogenous addition of ferritin and NADPH to yeast cell lysate. (B) Rates of iron release as a function of ferritin concentration. (C) Concentration of released iron, in one hour, as a function of ferritin, with % Fe(II) released shown in the inset. Conditions: 555 µL YCL, 1.5 mM NADPH, reaction volume 1 or 2.3 mL, ferritin concentration is indicated next to each kinetic trace, 3 mM ferrozine, 20 mM MOPS, pH 7.4. The red lines are first-order fits to the data, and the ferritin sample used here is recombinant human heteropolymer ferritin H₂₃L₁ loaded with 500 Fe/shell.

Figure 2

Iron reduction and release kinetics monitored by UV-Vis using cell lysate filtrate or retentate solutions separated using different MW membrane cut-offs (3, 10, 30, 50, or 100 kDa). (A) The control-subtracted iron release kinetics of the partitioned cell lysate solutions.(B) In the absence of exogenously added NADPH or ferritin, the iron release kinetics. (C) The kinetics of iron release were followed in the presence of 2–5 µM FMN, FAD, or riboflavin using freshly prepared whole cell lysate solutions. Conditions: 555 µL of the retentates or filtrates, 1.5 mM NADPH, 0.8 µM homopolymer H-ferritin or heteropolymer ferritins (H19:L5, H22:L2) loaded with 500Fe(III)/shell, 3 mM ferrozine, 2–5 µM FMN, FAD or riboflavin, 20 mM MOPS, pH 7.4. The displayed kinetics are averages of at least 2 different trials. The control kinetic experiment using cell lysate in the absence of ferritin is similar to that of buffer with ferritin.

Figure 3

(A) Iron release kinetics as a function of temperature. ((A), Inset) Differential scanning calorimetry of whole cell lysate. (B) Representative oxygen consumption kinetics of different YCL solutions (whole cell lysate, retentate, and filtrate from a 10 kDa membrane cut-off); control experiments of whole YCL with and without added ferritin are also included. (C) Example UV–vis absorption spectrum of a solution at the end of a kinetic run. All other experimental conditions are the same as those in Figure 1 and Figure 2.