Biophysical characterization of iron in mitochondria isolated from respiring and fermenting yeast

Abstract

The distributions of Fe in mitochondria isolated from respiring, respiro-fermenting, and fermenting yeast cells were determined with an integrative biophysical approach involving Mossbauer and electronic absorption spectroscopies, electron paramagnetic resonance, and inductively coupled plasma emission mass spectrometry. Approximately 40% of the Fe in mitochondria from respiring cells was present in respiration-related proteins. The concentration and distribution of Fe in respiro-fermenting mitochondria, where both respiration and fermentation occur concurrently, were similar to those of respiring mitochondria. The concentration of Fe in fermenting mitochondria was also similar, but the distribution differed dramatically. Here, levels of respiration-related Fe-containing proteins were diminished approximately 3-fold, while non-heme HS Fe(II) species, non-heme mononuclear HS Fe(III), and Fe(III) nanoparticles dominated. These changes were rationalized by a model in which the pool of non-heme HS Fe(II) ions serves as feedstock for Fe-S cluster and heme biosynthesis. The integrative approach enabled us to estimate the concentration of respiration-related proteins.

Figure 1

Western blot of EGTA-washed isolated mitochondria (left lane) and corresponding extract (right lane) from respiring cells (Batch R5). In both cases 60 μg of protein were loaded into the wells of a 10% SDS-PAGE gel. Kar2 is an endoplasmic reticular protein (5× increased in isolated mitochondria vs. cell extract); CPY is a vacuolar protein (6× decreased); PGK is a cytosolic protein (5× decreased); Porin is a mitochondrial protein (10× increased).

Figure 2

Mössbauer Spectra of packed mitochondria (batch R1) isolated from respiring cells. A, 4.5 K, 0.05 T field applied parallel to the γ radiation. The black line is a simulation for the sum of the central doublet, HS Fe^II hemes (dashed), and S = ½ [Fe₂S₂]¹⁺ clusters. See Table 1 and bar graph (Figure 7) for percentages for all components. B, same as A after subtracting the central doublet and HS Fe^II heme contributions. The purple solid line is a simulation for S = ½ [Fe₂S₂]¹⁺ clusters, while the black solid line is a composite simulation including these species and HS Fe^II hemes. The absorption between 0 and + 1 mm/s that is not covered by the black curve is unassigned. C, same as A but recorded at 100 K. The blue line outlines the contribution of the central doublet in the sample. D, same as A except with 8.0 T parallel applied field. The black line is a simulation that includes the central doublet and contributions from S = ½ [Fe₂S₂]¹⁺ clusters.

Figure 3

Electronic Absorption spectra of mitochondrial suspensions. A, respiring (R1); B, respiro-fermenting (RF2); C, fermenting (F3). Effective absorbances of neat mitochondria normalized to a 10 mm pathlength cuvette are plotted. These values were obtained by multiplying raw absorbances by 2.0 (the dilution factor relative to packed mitochondria) and 5.0 (pathlength factor due to the use of a 2 mm pathlength cuvette), and by dividing by 0.82 (the packing factor). Dashed lines are composites from individual heme a, b, and c containing proteins, using parameters given in Table S1 (averages in Table 1).

Figure 4

10 K EPR spectra of mitochondria from respiring (A, batch R2, decomposition in S5), respiro-fermenting (B, RF1), and fermenting (C, F11) cells. Spectra A and C were collected at 0.05 mW, spectrum B at 0.2 mW. Dashed lines are simulations, with batch-averaged parameters given in Table 1. D, E and F are the low-field regions of A, B and C, respectively.

Figure 5

Mössbauer spectra of packed mitochondria (RF1) from respiro-fermenting cells. A, Spectrum measured at 4.5 K, 0.05 T parallel applied field. The black line is a simulation for the central doublet, HS Fe^II hemes, NHHS Fe^II, and S = ½ [Fe₂S₂]¹⁺ clusters. The lines above the spectrum are simulations for S = ½ [Fe₂S₂]¹⁺ (purple), nonheme HS Fe^II (red) and the HS Fe^II hemes (black dashed line). B, same as A but at 100 K. C, same as A but at 8.0 T. The black line is a simulation for the central doublet and S = ½ [Fe₂S₂]¹⁺ clusters. The cyan line above is a simulation for HS mononuclear Fe^III species.

Figure 6

Mössbauer spectra of mitochondria (batch F9) from fermenting cells. A, 4.5 K, 0.05 T parallel applied magnetic field. The black line is a simulation for the sum of the central doublet, HS Fe^II hemes, NHHS Fe^II (high-energy line indicated by arrow), and the Fe^III nanoparticles. B, same as A except at 100 K. The red line indicates HS Fe^II; green line indicates nanoparticle contribution. C, same as A except at 8.0 T. The cyan line is a simulation for the HS Fe^III.

Figure 7

Bar graph showing the major forms of Fe present in respiring, respiro-fermenting, and fermenting mitochondria. Color-coding is matched to simulated features in previous figures.

Figure 8

Model describing the shift in the iron content of mitochondria with metabolic growth mode. The size of the NHHS Fe^II pool is dictated by the balance of input and output fluxes. During respiration, the pool is small (~ 15 μM). When cells ferment, the rate of Fe/S cluster and heme biosynthesis declines, causing the pool to enlarge (~ 150 μM). The rate of Fe^II import from the cytosol is not significantly affected by the change in metabolism. Under fermenting conditions, a portion of the NHHS Fe^II pool may become oxidized to mononuclear nonheme HS Fe^III, a subset of which may precipitate as Fe^III nanoparticles.