VSports最新版本 - Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official VSports app下载. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure V体育官网. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. .

. 2010 Jul 6;49(26):5436-44.
doi: 10.1021/bi100558z.

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

Jessica Garber Morales  1 , Gregory P Holmes-Hampton, Ren Miao, Yisong Guo, Eckard Münck, Paul A Lindahl
Affiliations

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

Jessica Garber Morales et al. Biochemistry. .

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 VSports手机版. The integrative approach enabled us to estimate the concentration of respiration-related proteins. .

PubMed Disclaimer

Figures

Figure 1
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
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 FeII 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 FeII 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 FeII 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
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
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
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 FeII hemes, NHHS FeII, and S = ½ [Fe2S2]1+ clusters. The lines above the spectrum are simulations for S = ½ [Fe2S2]1+ (purple), nonheme HS FeII (red) and the HS FeII 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 FeIII species.
Figure 6
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 FeII hemes, NHHS FeII (high-energy line indicated by arrow), and the FeIII nanoparticles. B, same as A except at 100 K. The red line indicates HS FeII; green line indicates nanoparticle contribution. C, same as A except at 8.0 T. The cyan line is a simulation for the HS FeIII.
Figure 7
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
Figure 8
Model describing the shift in the iron content of mitochondria with metabolic growth mode. The size of the NHHS FeII 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 FeII import from the cytosol is not significantly affected by the change in metabolism. Under fermenting conditions, a portion of the NHHS FeII pool may become oxidized to mononuclear nonheme HS FeIII, a subset of which may precipitate as FeIII nanoparticles.
See this image and copyright information in PMC

References (V体育官网入口)

    1. Hudder BN, Morales JG, Stubna A, Münck E, Hendrich MP, Lindahl PA. Electron paramagnetic resonance and Mössbauer spectroscopy of intact mitochondria from respiring Saccharomyces cerevisiae. Journal of Biological Inorganic Chemistry. 2007;12:1029–1053. - PubMed (VSports手机版)
    1. Lindahl PA, Morales JG, Miao R, Holmes-Hampton G. Chapter 15 Isolation of Saccharomyces cerevisiae mitochondria for Mössbauer, EPR, and electronic absorption spectroscopic analyses. Methods Enzymol. 2009;456:267–285. - PubMed
    1. Lill R, Muhlenhoff U. Maturation of iron-sulfur proteins in eukaryotes: Mechanisms, connected processes, and diseases. Annu Rev Biochem. 2008;77:669–700. - PubMed
    1. Beinert H. Spectroscopy of succinate dehydrogenases, a historical perspective. Biochimica Et Biophysica Acta-Bioenergetics. 2002;1553:7–22. - V体育安卓版 - PubMed
    1. Hunte C, Koepke J, Lange C, Rossmanith T, Michel H. Structure at 2.3 angstrom resolution of the cytochrome bc(1) complex from the yeast Saccharomyces cerevisiae co-crystallized with an antibody Fv fragment. Structure. 2000;8:669–684. - "V体育官网" PubMed

Publication types