Ferric ions accumulate in the walls of metabolically inactivating Saccharomyces cerevisiae cells and are reductively mobilized during reactivation

Abstract

Mössbauer and EPR spectra of fermenting yeast cells before and after cell wall (CW) digestion revealed that CWs accumulated iron as cells transitioned from exponential to post-exponential growth. Most CW iron was mononuclear nonheme high-spin (NHHS) Fe(III), some was diamagnetic and some was superparamagnetic. A significant portion of CW Fe was removable by EDTA. Simulations using an ordinary-differential-equations-based model suggested that cells accumulate Fe as they become metabolically inactive. When dormant Fe-loaded cells were metabolically reactivated in Fe-deficient bathophenanthroline disulfonate (BPS)-treated medium, they grew using Fe that had been mobilized from their CWs AND using trace amounts of Fe in the Fe-deficient medium. When grown in Fe-deficient medium, Fe-starved cells contained the lowest cellular Fe concentrations reported for a eukaryotic cell. During metabolic reactivation of Fe-loaded dormant cells, Fe(III) ions in the CWs of these cells were mobilized by reduction to Fe(II), followed by release from the CW and reimport into the cell. BPS short-circuited this process by chelating mobilized and released Fe(II) ions before reimport; the resulting Fe(II)(BPS)3 complex adsorbed on the cell surface. NHHS Fe(II) ions appeared transiently during mobilization, suggesting that these ions were intermediates in this process. In the presence of chelators and at high pH, metabolically inactive cells leached CW Fe; this phenomenon probably differs from metabolic mobilization. The iron regulon, as reported by Fet3p levels, was not expressed during post-exponential conditions; Fet3p was maximally expressed in exponentially growing cells. Decreased expression of the iron regulon and metabolic decline combine to promote CW Fe accumulation.

Figure 1. Low field (0.05 T) low temperature (5 K) Mössbauer spectra of whole intact fermenting WT cells grown on ⁵⁷Fe₄₀B₀ medium and harvested at different growth stages

A, harvested at OD₆₀₀ = 0.4 (exponential stage) and washed with water; B, harvested at OD₆₀₀ = 0.7 (exponential stage) and washed with water; C, same as B except treated with lyticase/EDTA; D, black hash-marks, harvested at OD₆₀₀ = 1.5 (post-exponential stage; 5 days) and water washed; E, same as D except washed with EDTA; F, same as D and E except treated with lyticase/EDTA. Red and blue hashmarks in D are the same spectra as shown in E and F, respectively, scaled to the spectrum in black. Solid red line in A is a simulation consisting of NHHS S = 5/2 Fe^III from vacuoles (green line; 74%; δ = 0.41 mm/s; ΔE_Q = 0.3 mm/s; A_iso/g_nβ_n = -228 kG; D = 0.5 cm^-1; E/D = 0.33; η = 3; Γ = 0.7 mm/s), HS Heme Fe^II (orange line: δ = 0.8 mm/s; ΔE_Q = 2.4 mm/s; Γ = 0.3 mm/s), central doublet (gold line: δ = 0.45 mm/s; ΔE_Q = 1.15 mm/s; Γ = 0.7 mm/s), and NHHS Fe^II (purple line: δ = 1.26 mm/s; ΔE_Q = 3.0 mm/s; Γ = 0.6 mm/s). The Y-axis scale for A, B, and C are the same.

Figure 2. 10 K X-Band EPR spectra of whole intact fermenting yeast cells

A, treated with water; B, treated with EDTA; C, treated with EDTA/lyticase. Other EPR conditions: microwave power, 0.2 mW; microwave frequency, 9.645 GHz; modulation amplitude, 9.2 G; sweep time, 160 sec. Samples were the same as those used to generate Figure 1 D, E, and F, respectively, obtained by transferring samples from MB holder to EPR tubes while maintained near 77 K.

Figure 3. Model of cell growth and O₂ consumption in a batch culture of WT fermenting S. cerevisiae

Upper panel: plots of OD₆₀₀ and [O₂] in ⁵⁷Fe₄₀B₀ medium vs. time after inoculation. Data are solid green (OD₆₀₀) and red (O₂) circles. Simulations are green (total cells), yellow (active cells), black (dormant cells), blue (nutrient concentration) and red (O₂ concentration) lines. Lower panel: chemical model showing generation of O₂, consumption of O₂ by active cells, self-replication of active cells, and interconversion of active and dormant cells as regulated by the nutrient concentration.

Figure 4. Temperature-dependent Mössbauer spectra of ⁵⁷Fe-loaded cells (water washed)

A, 5; B, 15; C, 25; D, 50; E, 75; F, 100; G, 150 (all in K). A field of 0.05 T was applied parallel to the gamma radiation. The sample used was the same as in Figure 1D. Overall simulations (red lines in A and F) were the sum of two simulated spectra, including an S = 5/2 species (A_iso/g_nβ_n = -216 kG, D = 0.04 cm^-1, E/D = 0.22, δ = 0.45 mm/s, ΔE_Q = 0.5 mm/s, η = 10 and Γ = 0.4 – 0.7 mm/s) representing 70% of spectral intensity, and an S = 0 species (δ = 0.38 mm/s, ΔE_Q = 0.5 mm/s, η = 1 and Γ = 0.4 – 0.7 mm/s) representing 30% of spectral intensity. With increasing temperature, spectral features broadened.

Figure 5. Variable-Field 4.2 K Mössbauer spectra of ⁵⁷Fe-loaded (water washed) cells

A, 0; B, 0.75; C, 1.5; D, 3.0, and E, 6.0 (all in T). Fields were applied perpendicular to the gamma radiation. Overall simulations (red lines) were generated using the same model as in the Figure 4 legend. The sample was the same as used in Figures 1D and 4. The simulation in A assumed an applied field of 0.02 T and that the sextet represented 65% of spectral intensity and the doublet 35%.

Figure 6. Temperature-dependent EPR spectra of ⁵⁷Fe-loaded cells (water washed)

Low-field spectra show the g = 4.3 signal while high-field spectra exhibit a broad g = 2 signal. Spectra were collected at 10 K (red line), 20 K (yellow), 40 K (green), and 80 K (blue). Signal intensities have been multiplied by temperature. The sample used was the same as in Figure 1D, 4 and 5. Microwave power was 2 mW and 0.2 mW for the low- and high-field spectra, respectively. Other conditions were as in Figure 2.

Figure 7. Iron concentrations in WT post-exponential cells and the corresponding washes after various treatments

A, [Fe]_cell; B, [Fe]_wash. Data are solid circles. Black, washed with water only; Red, washed with EDTA only; Blue, washed with lyticase and EDTA.

Figure 8. Panel A, Growth of Fe-loaded or Fe-starved Cells after transfer to various media

Solid circles, Fe-loaded cells transferred to Fe₀B₃₀ medium. Open circles, Fe-starved cells transferred to Fe₀B₃₀ medium. Solid triangles, Fe-loaded cells transferred to Fe₀B₁₀₀. Open triangles, Fe-starved cells transferred to Fe₀B₁₀₀ medium. Panel B, Growth of Fe-loaded cells transferred to various media inoculated at conserved cell density. Solid squares, transferred to ⁵⁷Fe₄₀B₀ medium. Open squares, transferred to Fe₀B₁₀₀ medium. Crosses, transferred to Fe₀B₁₀₀-NAB medium. Panel C, same as Panel B, but a different experiment. Solid squares, transferred to ⁵⁶Fe₄₀B₀ medium. Open squares, transferred to Fe₀B₁₀₀ medium. Solid diamonds, transferred to Fe₀B₀ medium. Open diamonds, transferred to DW.

Figure 9. Mössbauer spectra (5 K, 0.05 T) of whole WT yeast cells before (A) and after (B – E) switching growth media

Results from this experiment are also presented in Figure 8B. A, Fe-loaded cells. The blue line is a simulation of Fe^III nanoparticles (δ = 0.53 mm/s; ΔE_Q = 0.52 mm/s; Γ = 0.45 mm/s). The red line is a composite simulation including 20% absorption due to nanoparticles and 65% to NHHS Fe^III (A_iso/g_nβ_n = -235 kG; E/D = 0.33; D = 1.15 cm^-1; δ = 0.55 mm/s; ΔE_Q = 0 mm/s; Γ = 0.8 mm/s). B, Fe-loaded cells 5 days after being transferred to Fe₀B₁₀₀. Red line is a composite simulation including NHHS Fe^III and the quadrupole doublet due to ⁵⁷Fe^II(BPS)₃. Simulation parameters are given in Table S3. C, same as B but after removing the quadrupole doublet due to ⁵⁷Fe^II(BPS)₃. The green line is a simulation of the NHHS Fe^II doublet and the red line is a composite simulation defined in Table S3. D, Fe-loaded cells 1 day after being transferred to Fe₀B₁₀₀-NAB medium. Red line is a composite simulation. E, same as D but after removing the quadrupole double due to Fe^II(BPS)₃. Green and maroon lines are simulations of the two NHHS Fe^II species described in the text. The red line is a composite simulation.

Figure 10. Iron concentrations in growth medium before and after inoculation with ⁵⁷Fe-loaded cells

Other results of this experiment are presented in Figure 1C. Panel A, ⁵⁷Fe concentration in media: Solid squares, medium before and after ⁵⁷Fe-loaded cells were transferred to Fe₀B₁₀₀ medium. The datum at t < 0 was of medium prior to inoculation. Other data are of the medium 0, 6, 12 and 24 hr after the transfer and after cells were removed by centrifugation. Solid triangles, same but for Fe₀B₀ medium. Blank circles, same but for DW. Panel B, Fe concentration in ⁵⁶Fe₄₀B₀ medium: Solid circles, total [Fe]; Open diamonds, [⁵⁶Fe]; Solid diamonds [⁵⁷Fe].

Figure 11. Whole-cell Mössbauer spectra (5 K, 0.05 T) obtained from the experiment of Figure 8C

A, ⁵⁷Fe-loaded cells; B – F, ⁵⁷Fe-loaded cells after 1 day incubation in the following media: B, Fe₀B₀; C, DW; D, Fe₀B₁₀₀; E, same as D, but cells were rinsed with 100 mM Tris-HCl buffer (pH 9.4) three times prior to obtaining the spectrum; F, ⁵⁶Fe₄₀B₀. Red and blue lines in A and C are the same as in Figure 9A. The green line in B simulates the CD while the red line is a composite simulation as defined in Table S3. The red line in D simulates the Fe^II(BPS)₃ doublet, the green line in E simulates the CD, and the orange line in F simulates the NHHS Fe^II doublet.

Figure 12. Mössbauer spectra (5 K, 0.05 T) of ⁵⁷Fe-loaded cells before (A) and at increasing times after (B – E) transfer to Fe₀B₁₀₀ medium

Time after transfer: B, 30 min; C, 3 hr; D, 6 hr. Blue and pink lines are simulations of the Fe^III nanoparticle and Fe^II(BPS)₃ quadrupole doublets, respectively. The red lines are composite simulations as defined in Table S3. The arrow in B indicates the position of the high energy line of the NHHS Fe^II doublet.

Figure 13. Western Blot showing Fet3p expression levels in WT cells in various media

Top panel, Fe-loaded cells transferred to Fe₄₀B₀, ⁵⁶Fe₄₀B₀, and Fe₀B₀ media and harvested at the indicated times (in hr) after the transfer. Bottom panel, same as top panel but with cells transferred to Fe₀B₁₀₀ and DW media.

Figure 14. Model for iron accumulation into the cell wall

Metabolically active (exponentially-growing) cells do not accumulate Fe in their CWs; Fe^III from the environment is reduced by metabolic processes in the cell and it enters the cell as Fe^II. As cells transition into a post-exponential (or dormant) state, Fe^III begins to accumulate in the CW. CW Fe can be removed by chelation or CW digestion. When dormant cells become metabolically active (by placing them into fresh media), the CW Fe^III becomes reductively mobilized to the Fe^II state. The Fe^II is released from the CW where it can: a) dissociate from the cell and diffuse into the environment; b) enter the cytosol to support cell growth; or c) chelate with BPS (if BPS is in the medium). A significant portion of the neutral Fe^II(BPS)₃ species adsorbs onto the CW.