Electron paramagnetic resonance and Mössbauer spectroscopy of intact mitochondria from respiring Saccharomyces cerevisiae

J Biol Inorg Chem (2007) 12:1029 1053 DOI 10.1007/s00775-007-0275-1 ORIGINAL PAPER Electron paramagnetic resonance and Mössbauer spectroscopy of intact mitochondria from respiring Saccharomyces cerevisiae Brandon N. Hudder Æ Jessica Garber Morales Æ Audria Stubna Æ Eckard Münck Æ Michael P. Hendrich Æ Paul A. Lindahl Received: 5 April 2007 / Accepted: 27 June 2007 / Published online: 31 July 2007 Ó SBIC 2007 Abstract Mitochondria from respiring cells were isolated under anaerobic conditions. Microscopic images were largely devoid of contaminants, and samples consumed O 2 in an NADH-dependent manner. Protein and metal concentrations of packed mitochondria were determined, as was the percentage of external void volume. Samples were similarly packed into electron paramagnetic resonance tubes, either in the as-isolated state or after exposure to various reagents. Analyses revealed two signals originating from species that could be removed by chelation, including rhombic Fe 3+ (g = 4.3) and aqueous Mn 2+ ions (g = 2.00 with Mn-based hyperfine). Three S = 5/2 signals from Fe 3+ hemes were observed, probably arising from cytochrome c peroxidase and the a 3 :Cu b site of cytochrome c oxidase. Three Fe/Sbased signals were observed, with averaged g values of 1.94, 1.90 and 2.01. These probably arise, respectively, from the [Fe 2 S 2 ] + cluster of succinate dehydrogenase, the [Fe 2 S 2 ] + Electronic supplementary material The online version of this article (doi:10.1007/s00775-007-0275-1) contains supplementary material, which is available to authorized users. B. N. Hudder J. G. Morales P. A. Lindahl (&) Department of Chemistry, Texas A&M University, College Station, TX 77843-3255, USA e-mail: lindahl@mail.chem.tamu.edu A. Stubna E. Münck M. P. Hendrich Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213-2683, USA P. A. Lindahl Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA cluster of the Rieske protein of cytochrome bc 1, and the [Fe 3 S 4 ] + cluster of aconitase, homoaconitase or succinate dehydrogenase. Also observed was a low-intensity isotropic g = 2.00 signal arising from organic-based radicals, and a broad signal with g ave = 2.02. Mössbauer spectra of intact mitochondria were dominated by signals from Fe 4 S 4 clusters (60 85% of Fe). The major feature in as-isolated samples, and in samples treated with ethylenebis(oxyethylenenitrilo)tetraacetic acid, dithionite or O 2, was a quadrupole doublet with DE Q = 1.15 mm/s and d = 0.45 mm/s, assigned to [Fe 4 S 4 ] 2+ clusters. Substantial high-spin non-heme Fe 2+ (up to 20%) and Fe 3+ (up to 15%) species were observed. The distribution of Fe was qualitatively similar to that suggested by the mitochondrial proteome. Keywords Iron Sulfur Cluster assembly Heme biosynthesis Non-heme Abbreviations CoQ Coenzyme Q DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid EGTA Ethylenebis(oxyethylenenitrilo) tetraacetic acid EPR Electron paramagnetic resonance ETF Electron transfer flavoprotein HEPES N-(2-Hydroxyethyl)piperazine-N 0 - ethanesulfonic acid IM Inner membrane IMS Intermembrane space NHE Normal hydrogen electrode OM Outer membrane SH buffer 0.6 M sorbitol/20 mm N-(2-hydroxyethyl) piperazine-n 0 -ethanesulfonic acid buffer ph 7.4

1030 J Biol Inorg Chem (2007) 12:1029 1053 SP buffer Introduction 1.2 M sorbitol/20 mm potassium phosphate buffer ph 7.4 Mitochondria are the cellular organelles in which oxidative phosphorylation and a myriad of related processes involving iron, copper and manganese occur. These branched tubular structures have an outer membrane (OM), an aqueous intermembrane space (IMS), an inner membrane (IM) and an aqueous matrix region. The IM is highly invaginated, with cristae protruding into the aqueous matrix region. Imported iron ions are used in heme and iron sulfur (Fe/S) cluster biosynthesis. A portion of these nascent prosthetic groups are incorporated into mitochondrial apoproteins, while the remainder are exported to the cytosol. Imported copper and manganese ions are installed into cytochrome c oxidase and manganese superoxide dismutase, respectively. The proteins involved in these processes can be categorized in terms of the metal centers they contain. Proteins containing Fe 2 S 2, Fe 3 S 4 and/or Fe 4 S 4 clusters include succinate dehydrogenase [1 3], the Rieske protein [4], aconitase and homoaconitase [5, 6], ferredoxin/adrenodoxin [7 10], biotin synthase [11 15] and lipoic acid synthase [16 18]. Dihydroxyacid dehydratase catalyzes the dehydration of an intermediate in the biosynthesis pathway of branched-chain amino acids [19]. Although the metal center in this enzyme has not been well studied, the homologous enzyme from Escherichia coli contains an Fe 4 S 4 cluster [20]. Such clusters are also found in scaffold proteins which are used in the synthesis of Fe/S clusters, including Isu1p, Isu2p, Isa1p and Nfu1p [21 24]. A BLAST search suggests that the open reading frame YOR356W encodes the flavin adenine dinucleotide-containing and Fe 4 S 4 -containing electron transfer flavoprotein (ETF) dehydrogenase [25 27]. Other mitochondrial proteins contain heme groups. Heme b is found in cytochrome bc 1 [28], cytochrome c peroxidase [29], succinate dehydrogenase and flavocytochrome b 2 [30]. Heme a is found in cytochrome c oxidase [31], while heme c is contained in cytochrome c 1 and in both isoforms of cytochrome c [32]. Heme monooxygenase catalyzes the conversion of heme b to heme a within the heme biosynthetic pathway [33]. The homolog from E. coli contains heme b and heme a prosthetic groups [34 36]. When yeast cells are grown under respiratory conditions, the heme-b-containing catalase A (Cta1p) is targeted to the mitochondrial matrix [37]. Another group of proteins are involved in iron trafficking. Isa2p [38] and Yfh1p [39] help import Fe 2+ ions into the matrix and insert iron into ferrochelatase (Hem15p) for heme biosynthesis [40 42] and into scaffold proteins for Fe/S synthesis. IM proteins Mrs3p, Mrs4p, Mmt1p and Mmt2p carry iron into mitochondria [43, 44]. Heme O synthase (Cox10p) and heme A synthase (Cox15p) bind intermediate states of hemes [33, 35, 45]. Cytochrome c heme lyase (Cyc3p) and cytochrome c 1 heme lyase (Cyt2p) install heme c into cytochromes c and c 1, respectively [46]. Mdl1p exports heme groups [47], while Atm1p and Erv1p export Fe/S clusters [48, 49]. Coq7p is a yeast mitochondrial protein that contains a diiron center and serves as a monooxygenase/hydroxylase in coenzyme Q (CoQ) biosynthesis [50, 51]. Cytochrome c oxidase is the best-known copper-containing mitochondrial protein. The Cox1p subunit of this complex contains one copper ion (Cu B ) adjacent to heme a 3 in its active site, while the electron-transfer Cu A site in Cox2p contains two copper ions [52]. Cox23p, Cox17p, Sco1p and Cox11p are chaperones that import Cu ions into mitochondria and insert them into Cox1p and Cox2p during their assembly [53 55]. Copper ions in these chaperones are in the diamagnetic Cu + oxidation state. A small amount of the cytosolic copper-containing (Cu Zn) superoxide dismutase (Sod1p) appears to localize in the IMS of mitochondria [56]. Approximately 90% of mitochondrial copper is found in the matrix as a nonproteinaceously bound pool of Cu + ions [57]. Manganese superoxide dismutase (Sod2p) appears to be the only manganese-containing enzyme in Saccharomyces cerevisiae mitochondria. The mitochondrial manganese chaperone protein (Mtm1p) helps to import manganese ions and to install one of these ions into matrix-localized apo-sod2p [58]. Flavins and ubiquinone can be stabilized in an S = 1/2 semiquinone state that affords electron paramagnetic resonance (EPR) signals at the free-electron g value, 2.00. Flavin-containing mitochondrial proteins include a-ketoglutarate dehydrogenase [59], D-lactate cytochrome c oxidoreductases [60], glutathione reductase [61], thioredoxin reductase [62], glycerol-3-phosphate dehydrogenase [63], D-arabinono-1,4-lactone oxidase [64], acetolactate synthase [65], methylene tetrahydrofolate reductase [66], succinate dehydrogenase [67], Coq6p [68], Mmf1p [69] and ETF dehydrogenase [25 27]. The most important spectroscopic technique that has been applied to intact mitochondria is EPR, dating from the pioneering work of Beinert [70], who initially described high-spin heme signals from cytochrome c oxidase [71]. The g ave = 2.01 EPR signal from the inactivated [Fe 3 S 4 ] + form of aconitase was observed in crude intact rat-heart mitochondria exposed to H 2 O 2 [72]. EPR signals from the

J Biol Inorg Chem (2007) 12:1029 1053 1031 Rieske cluster of cytochrome bc 1 and the [Fe 2 S 2 ] + cluster of succinate dehydrogenase have also been observed in intact mitochondria [73 78]. EPR spectra of intact mitochondria were examined to determine the effect of abolishing heme biosynthesis on succinate dehydrogenase and the Rieske protein [75] and to determine the effects of Ca 2+ and Mn 2+ ions [79, 80]. Adrenodoxin levels in intact human placental mitochondria were examined by EPR [81]. Respiratory complexes in submitochondrial fractions have also been examined [82 84]. In contrast, there has been just one report of a Mössbauer spectrum of intact mitochondria, specifically of a strain in which yfh1 was deleted [39]. This genetic modification causes iron to accumulate in the matrix, and the observed Mössbauer spectral intensity exclusively reflected the accumulated iron. The control Mössbauer spectrum of wild-type mitochondria was devoid of any signals. This overview highlights the complexity of transition metal metabolism occurring within these organelles. We report on our efforts to establish a few simple yet unestablished aspects of iron metabolism in yeast mitochondria, namely, the absolute concentration of iron and of overall protein contained therein, and the proportion of that iron present in various types of centers (e.g., hemes, Fe/S clusters, etc.). Our approach was to investigate mitochondria from S. cerevisiae using EPR and Mössbauer spectroscopy along with various bioanalytical characterizations. For the first time using whole mitochondria, the absolute spin concentrations of detectable metal protein species have been quantified from EPR spectra. We investigated intact mitochondria prepared under different redox and/or isolation conditions. We determined the proportion of excluded buffer in these packed samples, which, when combined with metal and protein determinations of the packed samples, allowed us to estimate the absolute iron concentration contained within these organelles. This information, when combined with our spectroscopic results, allowed us to estimate, albeit in broad terms, how iron is distributed within the organelle. This distribution was then compared with that calculated from the ironcontaining proteins known to be present in the mitochondrial proteome. Materials and methods Cell growth and isolation of mitochrondia S. cerevisiae cells (strain D273-10B) were grown on SSlac medium (0.3% glucose, 1.7% lactate) [72 g yeast extract, 25 g ammonium chloride, 25 g potassium hydrogen phosphate, 12.5 g NaCl, 12.5 g CaCl 2, 14.4 g MgCl 2, 12.5 g glucose and 0.7 L of 60% sodium lacate syrup (Fisher) in 25-L solution] in a custom-built thermostatically controlled autoclavable 25-L glass fermenter in which cultures were stirred and bubbled with pure O 2 at a rate of approximately 3 L/min, dispersed through a fine glass frit with a diameter of 5 cm. Under these growth conditions, cells ferment on glucose at early stages of growth and then switch to respiration on lactate once the glucose has been consumed. Harvesting commenced when the optical density of a 1-cm solution at 600 nm reached 1.2 1.4. The culture was chilled to 278 K and harvested at 5,000 rpm using a Sorvall SLC-6000 rotor and a Sorvall Evolution centrifuge. Immediately after harvesting and without freezing the pelleted cells, mitochondria were isolated essentially as described in [85] except that all steps were performed anaerobically. Cell paste (100 150 g) was transferred into a refrigerated argon-atmosphere glove box (M. Braun) maintained at approximately 278 K and approximately 1 ppm O 2 as monitored continuously using a model 310 Teledyne analyzer. Buffers used in the isolation were degassed on a Schlenk line. For some preparations, all isolation buffers were supplemented with ethylenediaminetetraacetic acid (EDTA) or ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) (Acros) at final concentrations of 1 or 10 mm. In other preparations, no metal chelators were included. Cell paste was suspended in a 100 mm tris(hydroxymethyl)aminomethane sulfate/ 10 mm dithiothreitol (DTT) buffer (500 ml) and then spun at 5,000 rpm for 5 min in the SLC-6000 rotor. Subsequent centrifugations were performed under these conditions unless otherwise stated. The resulting pellet was suspended in 1.2 M sorbitol/20 mm potassium phosphate buffer, ph 7.4 (500 ml), hereafter referred to as SP buffer, using a rubber policeman. The resulting suspension was centrifuged, resuspended in SP buffer (500 ml), centrifuged again, and resuspended again in the same buffer. Cell walls were disrupted by adding 3 mg of 100 units/mg yeast lytic enzyme (Sigma) per gram of cell paste. The resulting spheroplasts were centrifuged, suspended in SP buffer (500 ml) and then centrifuged. The pellet was resuspended in 250 ml of 1.2 M sorbitol/40 mm N-(2-hydroxyethyl) piperazine-n 0 -ethanesulfonic acid (HEPES) ph 7.4 and 250 ml of 1 mm phenylmethylsulfonyl fluoride in doubledistilled H 2 O. The mixture was homogenized using 25 strokes of a 40-mL Dounce homogenizer (Fisher Scientific) during a period of 2 4 min. The suspension was centrifuged at 2,500 rpm for 5 min, and the supernatant was transferred to a fresh centrifuge bottle and centrifuged again under the same conditions. The supernatant, which consisted of crude mitochondria, was then centrifuged at 10,000 rpm in a Sorvall SLA-1500 rotor for 10 min. The resulting pellet was resuspended in 200 ml of a 0.6 M sorbitol/20 mm HEPES buffer ph 7.4, hereafter referred to as SH buffer. The resulting solution was centrifuged three

1032 J Biol Inorg Chem (2007) 12:1029 1053 more times, in the manner described in the previous three sentences, and the final pellet of crude mitochondria was resuspended in 20 ml SH buffer. This solution was loaded onto a discontinuous gradient solution composed of 10 ml of 15% and 10 ml of 20% (w/v) Histodenz 1 (Sigma) prepared in SH buffer and contained in Beckman Ultra Clear TM centrifuge tubes. The tubes were placed in the buckets of an SW-32Ti rotor (Beckman Coulter). The buckets were sealed, removed from the box and spun at 9,000 rpm in an SW-32Ti rotor (Beckman Coulter) for 1.5 h using a Beckman L7 ultracentrifuge. The buckets were returned to the box, and the tubes were placed in a support which allowed the pure mitochondrial band at the interface of the gradient to be collected after first removing the layer above the band. From 150 g cell paste, a total of 5 15 ml of mitochondrial solution in the as-isolated state was obtained using three to six buckets depending on the yield. The only reductant used during the procedure was DTT and then only at an early step of the isolation procedure before cell walls were disrupted. E 0 for the disulfide/dtt half-cell is 330 mv versus the normal hydrogen electrode (NHE) [86]. Anaerobically prepared isolation buffers undoubtedly contained a trace of oxidizing ability [87]. Both factors considered, the resulting solution potential of mitochondria in the non-redox-buffered as-isolated state was estimated to be between 0.1 and 0 mv versus NHE. Prior to freezing, some samples were exposed to air (typically for 1 day at 277 K), sodium dithionite (10 mm at ph 7.5 or 8.5), potassium ferricyanide (1 mm) or nitric oxide (1 atm). For Mössbauer spectroscopy studies, S. cerevisiae cells were grown similarly except that the medium was supplemented with 20 lm 57 FeCl 3. With use of a custommade Delrin TM insert that fit in the buckets of the SW-32Ti rotor, isolated mitochondria were packed tightly into Mössbauer cuvettes by centrifugation, typically at 9,000 rpm for 2 h. Samples were then frozen inside the glove box by contact with a liquid-nitrogen-cooled aluminum block. There was some variation in speed and duration used in packing, resulting in some differences in terms of observed 57 Fe concentrations. Each spectrum presented here was recorded with an approximately 40 mci 57 Co source. Electron and fluorescence microscopy One milliliter of as-isolated mitochondrial solution was microcentrifuged (Fisher Scientific) at 6,400 rpm for 5 min in a 1.5-mL Eppendorf tube. The pellet was resuspended in SH buffer and glutaraldehyde (2.0% v/v final concentration) was added. The solution was recentrifuged and the pellet was resuspended in 1% osmium tetroxide and 0.5% potassium ferrocyanide (w/v) in SH buffer. This was followed by en bloc staining using 1% uranyl acetate in SH buffer. Samples were dehydrated by incubation in increasingly concentrated ethanol solutions and then embedded using epoxy-based resin. Thin-sectioning was performed using a glass knife/water trough on a microtome, followed by retrieval of the thin sections using 200 mesh grids. Positive staining of these sections was performed using lead acetate/sodium hydroxide [88]. Images were obtained using a JEOL 1200 EX transmission electron microscope. For fluorescence images, equivalent mitochondrial solutions were incubated in SH buffer, containing 500 nm MitoTracker 1 (Molecular Probes) or, in another experiment, 1 lm ERTracker 1 at 310 K for 45 min. The solution was centrifuged, and the pellet was resuspended in SH buffer. Images were obtained using a Bio-Rad Radiance 2000 MP instrument equipped with a 63 (water-immersion) objective. Oxygen consumption measurements A sample of non-chelator-treated intact mitochondria was suspended in SH buffer. A 5-mL portion was assayed for protein concentration using the biuret method [89] as described in Protein and metal ion concentrations. Another portion of the intact mitochondrial solution was divided into three samples. One sample was incubated anaerobically for 4 5 h with 10 mm EDTA, another was incubated similarly with 10 mm EGTA, and the remaining sample was not treated. Each sample (1.2 ml) was injected into 29 ± 1 ml of air-saturated SH buffer containing 1.5 mm NADH, 0.2 mm ADP, 2 mm MgCl 2, 20 mm phosphates ph 7.4, 250 mm sucrose and 10 mm KCl, essentially as described in [90]. The solution was maintained at 298 K in a water-jacketed glass vessel which contained no gas head space. Included in this vessel was a Clark oxygen electrode (YSI Bioanalytical Products). The final protein concentration was 0.10 mg/ml. Electron paramagnetic resonance Custom-built Delrin TM inserts were designed to fit within the buckets of the SW-32Ti rotor. Holes were drilled into the center of these inserts, with a diameter just sufficient to fit a modified EPR tube (4.96-mm outer diameter; 3.39-mm inner diameter; 80-mm long; Wilmad/Lab Glass, Buena, NJ, USA). A 2-mm-long cylinder of silicone rubber was inserted at the bottom of the hole. The brown mitochondrial solution obtained from the gradient step described in Cell growth and isolation of mitochondria was diluted

J Biol Inorg Chem (2007) 12:1029 1053 1033 with an equal volume of SH buffer. Tubes were filled with this solution and the entire assembly was sealed, removed from the box and spun by centrifugation at 9,000 rpm for 1 h. Samples were returned to the box, and the supernatant was replaced with additional mitochondrial solution. This process was repeated until the volume of tightly packed mitochondria at the bottom of the tube reached approximately 400 ll. EPR tubes were removed from the inserts and frozen in less than 1 min using liquid N 2. Two to four EPR samples were prepared from a solution of gradientpurified mitochondria isolated from 25 L of culture. One end of a stainless steel wire (20 cm 0.5-mm diameter) was attached to one end of a stainless steel rod (20 cm 4.8-mm diameter), with the wire extended coaxially with the rod. Approximately 5 cm beyond the point of attachment, the wire was bent back towards the rod (like a hairpin) and coiled around itself up towards the rod. The outer diameter of the coil at the base of the hairpin was slightly less than the inner diameter of the modified EPR tube, while the outer diameter of the remainder of the coil was slightly greater than the inner diameter of the EPR tube. In this way, the wire coil fit snugly into the upper region of the EPR tubes. The entire assembly was just sufficiently robust to be inserted into and removed from the EPR cavity. Spectra were obtained with a Bruker EMX X- band EPR spectrometer operating in perpendicular mode with an Oxford Instruments EM910 cryostat. Signals were simulated with SpinCount written by one of the authors (M.P.H.). Signal intensities were quantified relative to a CuEDTA spin standard using the same software. Protein and metal ion concentrations A line was drawn on the exterior of the EPR tubes to indicate the height of the packed mitochondria. The organelles were thawed and quantitatively transferred to plastic screw-top vials using a slightly twisted quartz rod and a minimal volume of SH buffer. The volume of packed organelles was determined by weighing the tubes before and after filling them with an equivalent volume of water, and then dividing the difference by the density of water. The final volume of the solution in the screw-top vial, typically 5 ml, was similarly determined. The ratio of these two volumes constituted the dilution factor by which measured protein and metal concentrations, obtained using the solution in the vial, were multiplied to yield the respective concentrations in packed mitochondria. Samples contained in the vial were sonicated using a Branson Sonifier 450 operating for 5 10 min at 60% capacity. Protein analyses were performed in either of two ways, namely, by quantitative amino acid analysis, which is the most accurate method available [91], or by the biuret colorimetric method. Relative to amino acid analysis, the results obtained using the biuret method were similar within the uncertainty of the measurements. For quantitative amino acid analysis, aliquots were hydrolyzed in 6 M HCl/2% phenol at 383 K and analyzed using a Hewlett- Packard AminoQuant system. Amino acid percentages were similar among preparations. Primary and secondary amino acids present in the samples were derivatized using o-phthalaldehyde and 9-fluoromethylchloroformate, respectively. Metal concentrations were determined by atomic absorption spectrometry (PE AAnalyst 700 operating in furnace mode) and by inductively coupled plasma mass spectrometry (PerkinElmer). Sonicated samples (250 400 ll) were digested using an equal volume of 15.8 M trace-metal-grade HNO 3 (Fischer Scientific) in a sealed plastic tube that was then incubated overnight at 353 K. The resulting solution was diluted with deionized and distilled H 2 O to a final HNO 3 concentration of 0.2 M. Percentage of external solution in packed samples Custom-built Lexan graduated cylinders were constructed within inserts that fit within buckets of the SW- 32Ti rotor (Fig. 1). These inserts were used to accurately measure the volume of a packed mitochondria sample, obtained by loading a solution of isolated mitochondria and spinning the sample for 1 h at 9,000 rpm (10,000g). The supernatant was decanted and the volume of the packed sample was measured using this apparatus. This volume (V pel ) was assumed to be composed of the volume of the mitochondria plus the volume of excluded water: Fig. 1 Graduated cylinder used to measure the volume of packed mitochondria samples

1034 J Biol Inorg Chem (2007) 12:1029 1053 Table 1 Determination of excluded buffer in packed mitochondria samples * C stock (cpm/ml) V stock (ml) * C sup1 (cpm/ml) V sup1 (ml) V H2O 1 (ml) * C sup2 (cpm/ml) V sup2 (ml) V H2O 2 (ml) V pel (ml) Average %H 2 OInV pel 23,830 1.00 20,960 1.00 0.14 2,091 0.98 0.11 0.71 17 37,750 1.00 35,940 1.00 0.05 3,210 0.98 0.10 0.40 18 49,440 1.00 45,150 0.99 0.11 8,220 1.00 0.22 0.82 20 251,260 1.00 238,640 0.98 0.07 52,510 1.00 0.28 0.52 34 58,880 1.00 51,010 1.01 0.14 7,920 0.99 0.18 0.63 26 58,880 1.00 44,580 1.12 0.20 4,120 0.99 0.10 0.92 16 58,880 1.50 47,660 1.49 0.37 14,800 0.97 0.44 1.40 29 (V pel = V mito + V H2 O). To determine the ratio V H2 O/V pel,a 1.00-mL stock solution of radioactively labeled sucrose (American Radiolabeled Chemicals, 625 mci/mmol), prepared in SH buffer (with C stock in counts per minute per * milliliter given in Table 1 for each experiment), was added to the pellet and the pellet was resuspended. The inserts were spun by centrifugation as described above, the supernatant was removed, the volume (V sup1 ) was determined using a gastight syringe (Hamilton), and the concentration of radioactivity (C stock ) was determined by * scintillation counting (Beckman 5000SL). Assuming that none of the sucrose entered the mitochondria, the excluded water will also have a concentration of radioactivity given * by C stock. The conservation of matter suggests that C stock V stock ¼ C sup1 V sup1 þ C sup1 V H 2 O 1 : This equation was solved for V H2 O 1. The resulting pellet was found to have essentially the same volume as the original pellet. This pellet, containing radioactively labeled sucrose in the external volume, was resuspended with a solution of nonradioactively labeled sucrose, and the other steps of the same process were repeated. In this case, the resulting concentration of radioactivity in the supernatant * fraction was called C sup2 and the corresponding conservation of matter relationship becomes C sup1 V H 2 O 2 ¼ C sup2 V sup2 þ C sup2 V H 2 O 2 : This equation was solved for V H2 O 2. The average of the two values for V H2 O was divided by V pel, affording the fraction of the pellet volume due to excluded water. Results Characterization of intact mitochondria Intact yeast mitochondria were isolated as described in Materials and methods. Some preparations were isolated without adding a metal chelator to the isolation buffers, while others were isolated in the presence of either EDTA or EGTA. These chelators were added to remove adventitious metal ions associated with mitochondria. EGTA is unable to penetrate biological membranes [92], while this property is uncertain with respect to EDTA. However, EDTA has been used in isolating mitochondria [93] and as far as we are aware, there have been no reports of EDTA stripping essential metal ions from these organelles. We assayed a number of preparations for purity and membrane integrity using electron microscopy. Although significant size dispersion was typically evident (Fig. 2, top), there was no obvious evidence of impurities (bacteria or Golgi apparatus) or disrupted membrane structures. Sample morphology was independent of the method of isolation (as-isolated, EDTA-treated or EGTA-treated). Our images are similar to those obtained in the classical studies of Hackenbrock [94] and more recently [95]. Dispersion probably results from the dynamic fission and fusion processes that occur in yeast mitochondria [96]. Confocal microscopic images reveal that mitochondria form extensive tubelike networks extending throughout the cell [97]. These dynamic changes in size and shape would appear to render the concept of the number of mitochondria per cell rather meaningless. A more quantifiable parameter is the volume occupied by these organelles, and we will use this parameter throughout this paper. Fluorescence microscopy was also used to assess purity. One sample was stained for fluorescence with MitoTracker 1, while another was stained with ERTracker 1. The former dye associates with mitochondria, while the latter associates with the endoplasmic reticulum. As shown in Fig. 2, the vast majority of objects in our samples assimilated the MitoTracker 1 stain. There was no obvious sign of endoplasmic reticulum contamination using ERTracker 1 (data not shown). Both results suggest that the samples examined here were relatively pure and intact. We assayed a number of preparations for their ability to consume O 2. As shown in Fig. 3, preparations incubated in the absence of chelator or in the presence of EDTA or EGTA consumed 240, 160 and 200 nmol O 2 per minute per

J Biol Inorg Chem (2007) 12:1029 1053 1035 300 250 200 µ M Oxyge n 150 100 50 0 0 2 4 6 8 10 12 14 16 18 20 Time (min) Fig. 3 Oxygen consumption by isolated intact mitochondria. No chelator (squares), ethylenediaminetetraacetic acid (EDTA; triangles), ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA; circles). The experiment was performed as described in Materials and methods Fig. 2 Electron microscopy (top) and fluorescence microscopy (bottom) images of whole mitochondria isolated from Saccharomyces cerevisiae milligram of protein, respectively (estimated relative error of ±20%) when incubated in buffer containing NADH. Control samples assayed in the absence of NADH consumed little O 2. These rates are similar to those reported previously [98 100]. We also evaluated the coupling ratio of our preparations, defined as the rate of O 2 consumption with ADP added to the assay solution divided by the rate of consumption when ADP was absent. In our fresh samples, this ratio was approximately 2, similar to previously reported ratios [99, 100], whereas it approached 1 for mitochondria stored anaerobically at 278 K for approximately 3 days. Preparations used for EPR and Mössbauer analyses were frozen between 6 h and 3 days after they were isolated. We have not yet been able to correlate the age of the mitochondria to specific spectral changes, but we suspect that spectral features might become slightly broader with age. We determined protein and metal concentrations in our packed samples. The mean protein concentration (n = 15) was 55 ± 13 mg/ml, independent of whether chelators were or were not included in buffers during mitochondria isolations. In the absence of chelators, mean Fe, Cu, Mn and Zn concentrations in our packed mitochondria were 860 ± 480 lm (n = 5), 240 ± 150 lm (n = 5), 40 ± 30 lm (n = 5) and 1,000 ± 200 lm (n = 2), respectively. Corresponding metal concentrations for packed mitochondria samples isolated in the presence of chelators were 570 ± 100 lm Fe(n = 11), 220 ± 150 lm Cu(n = 11), 20 ± 10 lm Mn(n = 11) and 330 ± 170 lm Zn(n = 5). The scatter in the Cu, Mn and Zn data precludes us from drawing strong conclusions regarding the concentration of these ions in mitochondria. However, the modest scatter for the protein and Fe concentrations measured in samples isolated in the presence of chelators indicates that these concentrations (and their ratio, approximately 10 nmol Fe/mg protein) are reliable within a relative uncertainty of 25%. Next, we determined the proportion of the packed mitochondria samples due to the mitochondria themselves (rather than to excluded solution). Using the procedure described in Materials and methods, we found the percentage of mitochondria in our packed samples to be 77 ± 7 (n = 7), as shown in Table 1. The absolute concentrations of protein and Fe concentrations contained in neat mitochondria (devoid of solvent) could then be calculated by dividing the measured concentrations for the packed samples by 0.77, affording a protein concentration of approximately 70 mg/ml and Fe concentrations of 0.74 and 1.1 mm for samples isolated in the presence and absence of chelators, respectively. Given the uncertainty as

1036 J Biol Inorg Chem (2007) 12:1029 1053 to whether the Fe removed by chelators had any functional relevance, and on the basis of 57 Fe concentration estimates based on Mössbauer intensities (see Mössbauer spectra of mitochondria ), we conclude that the concentration of Fe in respiring yeast mitochondria is 800 ± 200 lm. EPR of mitochondria Mitochondria prepared in three different redox states, including as-isolated, oxidized and reduced, were packed tightly into custom-designed EPR tubes so as to expel external buffer and maximize the intensity of mitochondrial EPR signals. As-isolated samples are defined as those prepared anerobically in the absence of either oxidant or reductant. Oxidized samples were treated with either O 2 or ferricyanide. Reduced samples were treated with sodium dithionite. Some samples were prepared in the presence of the metal chelators EDTA and EGTA, while others were prepared in the absence of such chelators. This was done in an attempt to distinguish EPR signals originating from functional species within mitochondria from species that were adventitiously bound to the organelle. Owing to concern that membrane integrity would be compromised by freeze/thaw cycles, samples were never used twice (i.e., they were not thawed, treated in some manner, refrozen and reanalyzed spectroscopically). Once thawed, samples were used for protein and metal analyses and then discarded. This procedure produced reasonable but not perfect correlation between the redox state in which the sample was prepared and the types and intensities of EPR signals observed. EPR signals observed during this study are shown in Figs. 4 and 5. The principal g values for these signals and associated spin concentrations are compiled in Table 2. EPR signals of high-spin (S = 5/2) Fe 3+ species were analyzed with the conventional spin Hamiltonian H ¼ D½S 2 z 35=12 þ E=DðS2 x S2 y ÞŠ þ g 0bS B: For bb D it is customary to describe the magnetic properties of the three Kramers doublets by effective g values, which are dependent on the rhombicity parameter E/D and the intrinsic g value g 0 & 2.0 [101]. Mitochondria prepared in the absence of metal chelators sometimes exhibited a signal at g = 4.3, as shown in Fig. 4, spectrum A. This signal is typical of non-heme Fe 3+ species with E/D & 0.27 0.33. Such spectra also typically include features at g = 6.9 and 5.0, indicative of a highspin Fe 3+ heme with E/D = 0.041. The minor signal at g = 6.0 indicates a second high-spin Fe 3+ heme with E/D & 0 (discussed later). In such samples, the g =2 region (Fig. 5, spectrum A) is often dominated by a signal with a six-line hyperfine pattern (magnetic hyperfine coupling constant, a = 90 G) typical of an S = 5/2 Mn(II) species. A feature at g * 1.94 is also evident but is obscured by overlap with the Mn(II) signal. In all samples, the spectral region between g = 4.3 and 2.2 was devoid of signals. A more recently prepared sample, as-isolated in the absence of chelators, exhibited a g = 4.3 signal with significantly lower intensities than that in Fig. 4, spectrum A and did not show the Mn(II) signal of Fig. 5, spectrum A; rather it exhibited the spectrum shown in Fig. 5, spectrum B (discussed later). The g & 4.3 and Mn(II) signals were also either absent or present at low intensity in spectra of samples as-isolated with chelators included in all isolation buffers. This suggests that all or most of the species yielding these signals arise from adventitious Mn 2+ and Fe 3+ ions that can be chelated by EDTA and EGTA. g 8 7 6 5 4 A B C D E 6.9 6.4 6.0 5.4 5.0 80 100 120 140 160 180 200 B (mt) Fig. 4 Low-field X-band electron paramagnetic resonance (EPR) spectra of intact mitochondria. A Non-chelator-treated, as-isolated, B EGTA-treated, O 2 -exposed, C EGTA-treated, as-isolated, D EGTAtreated, reduced with 10 mm dithionite ph 7.4, E same as D but at ph 8.5. EPR conditions as follows: average microwave frequency, 9.45 GHz; microwave power, 20 mw; modulation amplitude, 10 G; receiver gain 1 10 4 for A C, 5.02 10 4 for D and E. Temperature, 10 K. The intensities of D and E have been multiplied by 5 and 2, respectively

J Biol Inorg Chem (2007) 12:1029 1053 1037 Fig. 5 High-field X-band EPR spectra of intact mitochondria. A Nonchelator-treated, as-isolated, B a more recent preparation of nonchelator-treated, as-isolated (200 lw), C EDTA treated, NO-exposed (9.458 GHz, 200 lw, gain 5.02 10 4 ), D EGTA-treated, O 2 - exposed, E EGTA-treated, oxidized with 1 mm ferricyanide. Other conditions were as for Fig. 4 except that the average microwave frequency was 9.43 GHz. The intensities of B E have been multiplied by 5, 5, 2, and 5, respectively. Microwave power in A, D, and E was 200 lw The same two high-spin heme species described above were also observed in spectra of chelator-treated as-isolated preparations, but an additional high-spin heme signal was also observed, with g = (6.4, 5.4) and E/D = 0.021. This signal was observed either alone (Fig. 4, spectrum B) or overlapped with the g = 6.0 feature (Fig. 4, spectrum C). The preparation affording the strong g = (6.4, 5.4) signal of Fig. 4, spectrum B had been exposed for approximately 20 min to O 2 prior to centrifugation and freezing under standard anaerobic conditions. In chelatortreated samples, the region between g = 4.3 and 2.2 was also devoid of signals. In general, the dominant signal in the g = 2 region from as-isolated chelator-treated samples had g = 2.026, 1.934 and 1.913 (g ave = 1.94), as in Fig. 5, spectrum B. The microwave power which caused the g ave = 1.94 signal intensity divided by the square root of the power to reach half maximum was P 1/2 = 57 mw at 10 K. On closer inspection, a second signal, with g = 2.02, 1.90 and 1.75 (g ave = 1.90) is also evident. The g values of the g ave = 1.94 and 1.90 signals strongly suggest that they arise from Fe/S proteins. An isotropic g iso = 2.00 signal was observed in most preparations. The signal was broader for some preparations (Fig. 5, spectrum B) and sharper in others (Fig. 5, spectrum D). A microwave power study at 10 K indicates that the sharp g iso = 2.00 signal is easily saturated at less than 1 lw. The other signals in the spectrum begin to saturate at powers greater than 80 lw. A fourth signal with one principal g value near 2.08 can also be observed in many preparations; however, the other associated g values are poorly resolved at 10 K. Spectral overlap became less problematic at 130 K, as this signal remains slow-relaxing, while the g ave = 1.94 and 1.90 signals are broadened at that temperature; spectra collected at that temperature suggest that the other features associated with the g = 2.08 resonance are near g = 1.99, affording Table 2 Electron paramagnetic resonance (EPR) signals observed from whole mitochondria from Saccharomyces cerevisiae Signal Spin state parameters g values (g 1, g 2, g 3 ) Concentration range (lm) Tentative assignment High-spin Fe 3+ heme 1 S = 5/2, E/D = 0.041 6.9, 5.0 0 3 Cytochrome c peroxidase (Ccp1p) High-spin Fe 3+ heme 2 S = 5/2, E/D = 0.021 6.4, 5.4 0 2 Cytochrome c oxidase, heme a 3 :Cu b High-spin Fe 3+ heme 3 S = 5/2, E/D = 0 6.0 0 1 Cytochrome c oxidase, heme a 3 :Cu b g = 4.3 S = 5/2, E/D = 0.33 4.27 Minor to 40 Adventitious Fe 3+ g ave = 2.02 S = 1/2 or spin-coupled system 2.085, 1.989, 1.985 1 20 Unassigned; possibly spin-interacting Fe/S clusters of ETF dehydrogenase g ave = 2.01 S = 1/2 2.026, 2.022, 2.003 0 5 [Fe 3 S 4 ] + probably from aconitase or succinate dehydrogenase g = 2.00 (hyperfine) S = 5/2; I = 5/2; a = 90 G 2.000, 2.000, 2.000 0 20 Adventitious Mn 2+ g = 2.00 (isotropic) S = 1/2 2.000, 2.000, 2.000 <2 C- or O-based organic radical g ave = 1.94 S = 1/2 2.026, 1.934, 1.913 0 23 Succinate dehydrogenase [Fe 2 S 2 ] + (Sdh2p) g ave = 1.90 S = 1/2 2.025, 1.897, 1.784 0 45 Rieske [Fe 2 S 2 ] + cluster (Rip1p) ETF electron transfer flavoprotein

1038 J Biol Inorg Chem (2007) 12:1029 1053 g ave = 2.02 for the signal. This was confirmed by spectral simulation and decomposition. The 10 K and 200 lw spectrum of an EGTA-treated sample was decomposed (Fig. 6, spectrum A, solid line) by simulating the g ave = 1.90 (Fig. 6, spectrum B), 1.94 (Fig. 6, spectrum C), 2.00 (Fig. 6, spectrum D) and 2.02 (Fig. 6, spectrum E) signals, using g values listed in Table 2. The intensity of each simulation was adjusted to produce a sum of the four simulations (Fig. 6, spectrum A, dashed line) that best matched the experimental spectrum. Experimental spectra from other preparations gave similar deconvolutions. EPR of intact mitochondria treated with various redox agents Earlier mitochondrial preparations that had been exposed to air for 1 2 days exhibited low-field regions essentially devoid of heme-containing signals. More recent preparations, exposed to O 2 for 6 h, exhibited the high-spin heme signal at g = (6.4, 5.4) at high concentration (Fig. 4, spectrum B). In these samples, the g = 2 region generally consisted of intense signals with g ave = 2.01 and g iso = 2.00, and were largely devoid of g ave = 1.94 and 1.90 signals (e.g., Fig. 5, spectrum D). A similar set of signals were observed in a sample oxidized with ferricyanide. In this case, the low-field region displayed a mixture of the g = 6.0 and g = (6.4, 5.3) high-spin heme signals, while the high-field region revealed an intense g ave = 2.01 signal (spin concentration approximately 5 lm) (Fig. 5, spectrum E) along with a sizable g iso = 2.00 signal (1 lm) and low-intensity g ave = 1.94 and 1.90 signals. The g ave = 2.01 signal was not observed in spectra of samples treated with dithionite or in spectra of most asisolated preparations, indicating that the species exhibiting this signal is EPR-silent when reduced. The low-field region of spectra from samples treated with dithionite was largely devoid of signals, as expected from the thermodynamic ability of dithionite to reduce Fe 3+ hemes. The g ave = 1.94 and 1.90 signals were present, as expected, but with concentrations similar to that observed in as-isolated samples. The reduction ability of dithionite declines as ph is lowered [102], and we anticipated that the intensity of these signals might increase significantly at ph 8.5 relative to the intensity at ph 7.4. This expectation was not fulfilled. However, an unresolved absorption-like feature at g * 6.4 was observed in spectra of a sample reduced with dithionite at ph 8.5 (Fig. 4, spectrum E) but not at ph 7.4 (Fig. 4, spectrum D). This may be associated with S = 3/2 [Fe 4 S 4 ] + clusters [103]. Another preparation was exposed to 1 atm NO. This afforded a signal at g \ = 2.07 and a g = 2.01 resonance that exhibited a 14 N hyperfine splitting of a = 14 G (Fig. 5, spectrum C). This signal is characteristic of a pentacoordinate heme nitrosyl complex [101]. The spin concentration associated with this signal (20 lm) was quite high, and it may reflect the overall concentration of pentacoordinate Fe 2+ heme species present, as such species are known to bind to NO to yield similar signals. Mössbauer spectra of mitochondria For readers not familiar with details of Mössbauer spectroscopy we have given a short tutorial-type section in the supplementary material. For the present study we have collected Mössbauer spectra from numerous samples of intact mitochondria. A spectrum from an as-isolated sample not exposed to metal chelators, shown in Fig. 7, spectrum A, exhibits three distinct spectral features (similar spectra were observed for preparations treated with metal chelators, EGTA and EDTA). Approximately 15 20% of the iron belongs to a doublet with quadrupole splitting DE Q & 3.3 mm/s and isomer shift d & 1.3 mm/s; this doublet is outlined in the experimental spectrum. The quoted values are typical of high-spin mononuclear Fe 2+ ions in penta- or hexacoordinate nitrogen/oxygen environments: Fe II (H 2 O) 6 complexes, the iron sites in reduced dioxygenases and iron superoxide dismutase, and of fully reduced binuclear iron-oxo centers at low applied field. High-spin Fe 2+ hemes have distinctly smaller d values (approximately 0.83 0.93 mm/s); however, such species would be difficult to resolve if they were to account for less than 5% of the Fe in the present samples. A second doublet in Fig. 7, spectrum A (outlined as the dashed line in Fig. 7, spectrum B), accounting for 55 65% of the iron, has DE Q & 1.15 mm/s and d & 0.46 mm/s. 1 This doublet most probably represents Fe 4 S 4 clusters in the 2+ core oxidation state. In this state [Fe 4 S 4 ] 2+ clusters have a ground state with cluster spin S = 0. Low-spin Fe 2+ hemes such as cytochromes b and c have very similar DE Q and d values and thus their contributions would be difficult to separate from those of [Fe 4 S 4 ] 2+ clusters. In principle, the cytochromes should be oxidizable and thus detectable by EPR; however, no such signals were identified in the analogous samples examined by EPR. Thus, we suspect that low-spin Fe 2+ hemes do not contribute substantially to 1 For a purified Fe 4 S 4 ferredoxin the area under the doublet can be quantified to within 1 2%. Here, the uncertainties are considerably larger, primarily because more than one cluster contributes. The primary contributors to the doublet may be aconitase and dihydroxyacid dehydratase. Because species with slightly different but unresolved parameters contribute, lineshapes are heterogeneously broadened Lorentzians. We used both the Lorentzian and the Voight lineshape options of WMOSS. As Voight shapes are narrower at the base, this option yields, upon visual inspection, a lower estimate for the concentration.

J Biol Inorg Chem (2007) 12:1029 1053 1039 0 A 1 0 B Absorption (%) 1 0.0 0.5 0 1 C D 8 T -5 0 5 Velocity (mm/s) Fig. 6 Simulations of the EPR spectrum of Fig. 5, spectrum B for asisolated mitochondria without chelator. A Data (solid line) and sum (dashed line) of simulations (B E). The g values of the simulations are stated for species with g ave and concentrations of B 1.90, 44 lm; C 1.94, 23 lm; D 2.00, 2 lm; E 2.02, 17 lm. Experimental conditions are the same as for Fig. 5, spectrum B this doublet. We comment further on the DE Q = 1.15 mm/s component when we discuss the spectrum of Fig. 7, spectrum C. A third component present in Fig. 7, spectrum A exhibits broad absorption extending over a velocity range of roughly 10 mm/s; this feature reflects unresolved magnetic hyperfine structure of (mostly) high-spin Fe 3+ ions as well as other unidentified low-spin magnetic species. Finally, as much as 12% of the total iron may belong to S = 1/2 [Fe 4 S 4 ] + clusters (discussed below). In principle, Mössbauer spectroscopy can be used, with some effort and proper calibration, to determine the absolute 57 Fe concentration of a sample. We have done this for many years, mainly for keeping track of 57 Fe enrichment in proteins, which we have calibrated with ferredoxins and Fig. 7 Low-temperature (4.2 K) Mössbauer spectra of intact asisolated mitochondria. A Spectrum of a preparation not treated with chelators, recorded in a 45 mt applied magnetic field. The spectrum of high-spin Fe 2+ components is outlined above the experimental data. B EGTA-treated mitochondria with magnetic field as for A. The dashed line indicates the DE Q = 1.15 mm/s doublet, a component comprising predominantly [Fe 4 S 4 ] 2+ clusters. The solid line represents the sum of the Fe 2+ and [Fe 4 S 4 ] 2+ components. C Same as for B but in the presence of a parallel applied magnetic field of 8.0 T. The dashed line is a spectral simulation generated under the assumption that the DE Q = 1.15 mm/s component is diamagnetic. The solid line above the data is a simulation for a high-spin Fe 3 component, and the solid line drawn through the data is the sum of the Fe 3+ and [Fe 4 S 4 ] 2+ species. The Fe 2+ component was not simulated because this would require use of many unknown parameters. D The 45-mT spectrum of mitochondria treated with O 2 /antimycin. The solid line outlines the contributions of the DE Q = 1.15 mm/s doublet (52%) and the Fe 2+ component (12%) dioxygenases. Thus, with our equipment, a 5-mm-thick frozen aqueous solution sample containing 1 mm 57 Fe exhibiting a quadrupole doublet of 0.30 mm/s full width at half maximum yields 5% resonance absorption. Using this empirical rule (comparing the total absorption area to that under the standard doublet), the sample of Fig. 7, spectrum A has a 57 Fe concentration of approximately 0.5 mm. A similarly prepared sample (Fig. 7, spectrum B), but treated with the chelator EGTA, has an 57 Fe concentration of approximately 0.3 mm (both calculations taking into effect the solvent void volume). Although unsupplemented with Fe, the media in which the yeast were grown certainly contained some natural-abundance Fe; thus, these Mössbauer spectroscopy-based estimates may be somewhat

1040 J Biol Inorg Chem (2007) 12:1029 1053 Table 3 Summary of Mössbauer and EPR results Fe center O 2 /antimycin As-isolated, no chelator As-isolated, EGTA Dithionite-treated EPR S = 0 [Fe 4 S 4 ] 2+ + low-spin Fe 2+ 52 57% 55 65% 40 50% 45% S = 1/2, 3/2 [Fe 4 S 4 ] + <8% <12% 40% to minor 6% (g ave = 2.02) S = 2, 1/2 [Fe 3 S 4 ] 0/+ <5% <5% <5% <5% 3% (g ave = 2.01) S = 0 [Fe 2 S 2 ] 2+ <5% ND ND ND S = 1/2 [Fe 2 S 2 ] + <12% <12% 10% High-spin Fe 3+, octahedral, N/O ligands 5% ND 15% 1% + adventitious High-spin Fe 2+ 5/6-CN, O/N ligands 12% 15 20% 20% 20% Low-spin Fe 3+ ND ND EGTA ethylenebis(oxyethylenenitrilo)tetraacetic acid, ND not determined lower than those obtained by chemical analysis since Mössbauer spectroscopy only detects 57 Fe in our samples. The EGTA-treated sample (Fig. 7, spectrum B) contains essentially the same spectral components as the sample that was not treated with chelators (Fig. 7, spectrum A); however, the proportions were somewhat different, with 40 50% in the DE Q = 1.15 mm/s doublet, approximately 20% high-spin Fe 2+, 15% high-spin Fe 3+ ions and some as yet unidentified iron. These percentages for the samples discussed in this study are summarized in Table 3. Figure 7, spectrum C was recorded at 4.2 K in the presence of an external magnetic field of 8.0 T applied parallel to the c beam. The central feature, outlined separately by the dashed line, belongs to the feature assigned to [Fe 4 S 4 ] 2+ clusters. This simulation was generated with the assumption that the DE Q = 1.15 mm/s doublet represents iron residing in a diamagnetic (S = 0) environment, in good agreement with the data. The two absorption bands at +8 and 8 mm/s Doppler velocity belong to high-spin Fe 3+ species with N/O octahedral coordination; a spectral simulation (solid line) is shown above the data. This component, representing approximately 15% of the 57 Fe, is probably a collection of various mononuclear Fe 3+ species with octahedral N/O ligation; such species typically have zero-field splitting parameters D < 2/cm and isotropic magnetic hyperfine coupling constants A 0 & (27 29) MHz. 2 Figure 8 shows 4.2 K Mössbauer spectra of mitochondria treated with 10 mm dithionite at ph 8.5; the spectra were recorded in parallel applied fields of 50 mt (Fig. 8, 2 In weak applied fields, the lowest three Kramers doublets of the spin sextet are generally populated at 4.2 K, yielding three Mössbauer spectra per site. Moreover, under these conditions the magnetic splittings, like the effective g values observed by EPR, are very sensitive to the rhombicity parameter E/D. Consequently, the highspin Fe 3+ ions in our sample produce broad and barely discernible features in weak fields. However, the 8.0-T spectra are fairly insensitive to D and E/D, because the large Zeeman splitting puts essentially all Fe 3+ ions into the M S = 5/2 state, facilitating detection and quantification. spectrum A) and 8.0 T (Fig. 8, spectrum B). For this sample approximately 45% of the 57 Fe is found to be associated with the DE Q = 1.15 mm/s doublet. Compared with the Fig. 7, spectrum A, there is increased absorption (from various paramagnetic species) around 1.8 and +2.2 mm/s Doppler velocity (arrows). These features most probably belong to S = 1/2 [Fe 4 S 4 ] + clusters. The solid lines overlapping the data in Fig. 8 are simulations typical of S = 0 [Fe 4 S 4 ] 2+ clusters (drawn as the dashed lines to represent 45% of total Fe) and from S = 1/2 [Fe 4 S 4 ] + (40%) clusters; the latter are, somewhat arbitrarily, represented by two cluster forms using the parameters of reduced aconitase (20%) and a (generic) set of parameters similar to those of the [Fe 4 S 4 ] + cluster E. coli sulfite reductase (20%). These two spectral components which have been used to represent the [Fe 4 S 4 ] + state could also be drawn into Fig. 7, spectrum A, each representing 10% of the 57 Fe in that spectrum. Interestingly, with the present decomposition, approximately 85% of the 57 Fe would belong to Fe 4 S 4 clusters in the dithionite-reduced sample. This estimate is probably a bit high, as some of the absorption attributed to [Fe 4 S 4 ] + clusters may result from [Fe 2 S 2 ] + clusters (but not more than 12%; see next paragraph). Additional absorption attributed to [Fe 4 S 4 ] 2+ clusters may also arise from low-spin Fe 2+ cytochromes. In a low external field, oxidized Fe 3 S 4 clusters would be present as an S = 1/2 absorption extending beyond the central [Fe 4 S 4 ] 2+ doublet. While we see no direct evidence for the presence of such species, they could be present at concentrations below 5% of the total 57 Fe. We attempted to oxidize anaerobically isolated mitochondrial samples by exposing them to air for 1 2 days. Such treatment had little effect on Mössbauer spectral features, and we suspected that this redox-buffering ability was related to the functioning of the respiratory electron transport chain. In an attempt to block this chain and thus prevent cytochrome oxidase from reducing O 2, we treated a sample with antimycin A, a potent inhibitor of cytochrome bc 1 [105], and then exposed it to O 2. The spectrum of this