Recovery of mrs3Δmrs4Δ Saccharomyces cerevisiae Cells under Iron-Sufficient Conditions and the Role of Fe580

Abstract

Mrs3 and Mrs4 are mitochondrial inner membrane proteins that deliver an unidentified cytosolic iron species into the matrix for use in iron-sulfur cluster (ISC) and heme biosynthesis. The Mrs3/4 double-deletion strain (ΔΔ) grew slowly in iron-deficient glycerol/ethanol medium but recovered to wild-type (WT) rates in iron-sufficient medium. ΔΔ cells grown under both iron-deficient and iron-sufficient respiring conditions acquired large amounts of iron relative to WT cells, indicating iron homeostatic dysregulation regardless of nutrient iron status. Biophysical spectroscopy (including Mössbauer, electron paramagnetic resonance, and electronic absorption) and bioanalytical methods (liquid chromatography with online inductively coupled plasma mass spectrometry detection) were used to characterize these phenotypes. Anaerobically isolated mitochondria contained a labile iron pool composed of a nonheme high-spin Fe^II complex with primarily O and N donor ligands, called Fe₅₈₀. Fe₅₈₀ likely serves as feedstock for ISC and heme biosynthesis. Mitochondria from respiring ΔΔ cells grown under iron-deficient conditions were devoid of Fe₅₈₀, ISCs, and hemes; most iron was present as Fe^III nanoparticles. O₂ likely penetrates the matrix of slow-growing poorly respiring iron-deficient ΔΔ cells and reacts with Fe₅₈₀ to form nanoparticles, thereby inhibiting ISC and heme biosynthesis. Mitochondria from iron-sufficient ΔΔ cells contained ISCs, hemes, and Fe₅₈₀ at concentrations comparable to those of WT mitochondria. The matrix of these mutant cells was probably sufficiently anaerobic to protect Fe₅₈₀ from degradation by O₂. An ∼1100 Da manganese complex, an ∼1200 Da zinc complex, and an ∼5000 Da copper species were also present in ΔΔ and WT mitochondrial flow-through solutions. No lower-mass copper complex was evident.

Figure 1.

Optical densities of growing WT (●) and ΔΔ (▲) cultures.

Figure 2.

Mössbauer spectra of whole cells (-C ending) and isolated mitochondria (-M ending). Solid lines are simulations for the central doublet (green), NHHS Fe^II doublet (blue), vacuolar high-spin Fe^III sextet (purple), Fe^III oxyhydroxide nanoparticles (gold), [Fe₂S₂]²⁺ doublet (teal), and high-spin Fe^II heme doublet (brown). The temperature was 5 K, and a field of 0.05 T was applied parallel to the γ rays. Solid red lines are composite simulations assuming the area percentages listed in Tables 1 and 2.

Figure 3.

Model explaining the Mrs3/4ΔΔ phenotype. Nutrient iron enters the cell and becomes cytosolic Fe^II. Cytosolic Fe^II can enter vacuoles where most of vacuolar Fe^II oxidizes to Fe^III. In WT cells, very little converts into nanoparticles. Some cytosolic Fe^II converts into cytosolic [Fe₄S₄]²⁺ clusters. The remaining cytosolic Fe^II enters WT mitochondria through the Mrs3/4 import pathway and through a slow alternative pathway, where it becomes the mitochondrial Fe^II pool. Cytosolic Fe^II can enter ΔΔ mitochondria only through the alternative pathway. Mitochondrial Fe^II is feedstock for the biosynthesis of ISCs and heme centers, the majority of which are installed in respiratory complexes that catalyze the reduction of O₂ to water. O₂ is constantly diffusing into the matrix. In healthy WT cells, the activity of the respiratory complexes is sufficiently high to prevent O₂ from diffusing in, but in iron-deficient ΔΔ cells, the activity is too low to prevent penetration. In that case, O₂ reacts with the Fe^II pool to generate mitochondrial nanoparticles. The cell responds by increasing the level of expression of iron importers on the plasma membrane. Under iron-sufficient conditions, the cytosolic Fe^II concentration is high, allowing sufficient iron to enter mitochondria and generate sufficient respiration activity to re-establish anaerobic conditions in the matrix. However, the size of the mitochondrial Fe^II pool and/or heme centers remains subnormal such that the iron regulon remains activated.

Figure 4.

Electronic absorption spectra of isolated anaerobic mitochondrial suspensions. Packed mitochondria were diluted 1:1 with buffer and transferred to a 2 mm path-length quartz cuvette; the cuvette was sealed with a stopper and removed from the box, and spectra were collected. We estimate a protein concentration of ~80 mg/mL in the sample based on previous results. Spectra have been offset for viewing.

Figure 5.

EPR spectra of ΔΔ and WT cells. The temperature for ΔΔ1_BPS, ΔΔ10, WT1_BPS, and WT10 spectra was 10 K, while that in others was 4.2 K; intensities were temperature-adjusted to allow comparisons. Other parameters: average microwave frequency, 9.373 ± 0.003 GHz; microwave power, 0.2 mW; modulation amplitude, 10 G; gain, 1000; conversion time, 0.3 s. Displayed intensities on the left were adjusted as indicated for ease of viewing. None of the spectra on the right side was adjusted.

Figure 6.

LC–ICP-MS chromatograms of LMM flow-through solutions prepared from the soluble fractions of WT and ΔΔ mitochondrial detergent extracts. The top panel shows ⁵⁶Fe detection. Trace intensities were adjusted as indicated for ease of viewing. The bottom panel shows ⁵⁶Fe, ³⁴S, and ³¹P detection of flow-through solutions from mitochondria harvested from WT40 cells harvested as cells were undergoing the transition to the stationary state. Traces collected immediately are labeled –0D, while those collected after a 5 day incubation are labeled –5D.

Figure 7.

Mn (top), Zn (middle), and Cu (bottom) LC–ICP-MS traces of flow-through solutions of soluble extracts of mitochondria isolated from WT and ΔΔ cells.