Electron Paramagnetic Resonance and Mo¨Ssbauer 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 Mo¨ssbauer spectroscopy of intact mitochondria from respiring Saccharomyces cerevisiae

Brandon N. Hudder Æ Jessica Garber Morales Æ Audria Stubna Æ Eckard Mu¨nck Æ Michael P. Hendrich Æ Paul A. Lindahl

Received: 5 April 2007 / Accepted: 27 June 2007 / Published online: 31 July 2007 SBIC 2007

Abstract Mitochondria from respiring cells were isolated cluster of the Rieske protein of cytochrome bc1, and the + under anaerobic conditions. Microscopic images were lar- [Fe3S4] cluster of aconitase, homoaconitase or succinate gely devoid of contaminants, and samples consumed O2 in dehydrogenase. Also observed was a low-intensity isotropic an NADH-dependent manner. Protein and metal concen- g = 2.00 signal arising from organic-based radicals, and a trations of packed mitochondria were determined, as was the broad signal with gave = 2.02. Mo¨ssbauer spectra of intact percentage of external void volume. Samples were similarly mitochondria were dominated by signals from Fe4S4 clusters packed into electron paramagnetic resonance tubes, either in (60–85% of Fe). The major feature in as-isolated samples, the as-isolated state or after exposure to various reagents. and in samples treated with ethylenebis(oxyethylenenitril-

Analyses revealed two signals originating from species that o)tetraacetic acid, dithionite or O2, was a quadrupole doublet 3+ could be removed by chelation, including rhombic Fe with DEQ = 1.15 mm/s and d = 0.45 mm/s, assigned to 2+ 2+ 2+ (g = 4.3) and aqueous Mn ions (g = 2.00 with Mn-based [Fe4S4] clusters. Substantial high-spin non-heme Fe hyperﬁne). Three S = 5/2 signals from Fe3+ hemes were (up to 20%) and Fe3+ (up to 15%) species were observed. observed, probably arising from cytochrome c peroxidase The distribution of Fe was qualitatively similar to that and the a3:Cub site of cytochrome c oxidase. Three Fe/S- suggested by the mitochondrial proteome. based signals were observed, with averaged g values of 1.94, 1.90 and 2.01. These probably arise, respectively, from the Keywords Iron Sulfur Cluster assembly + + [Fe2S2] cluster of succinate dehydrogenase, the [Fe2S2] Heme biosynthesis Non-heme

Abbreviations CoQ Coenzyme Q Electronic supplementary material The online version of this DTT Dithiothreitol article (doi:10.1007/s00775-007-0275-1) contains supplementary material, which is available to authorized users. EDTA Ethylenediaminetetraacetic acid EGTA Ethylenebis(oxyethylenenitrilo) B. N. Hudder J. G. Morales P. A. Lindahl (&) tetraacetic acid Department of Chemistry, EPR Electron paramagnetic resonance Texas A&M University, College Station, TX 77843-3255, USA ETF Electron transfer ﬂavoprotein 0 e-mail: [email protected] HEPES N-(2-Hydroxyethyl)piperazine-N - ethanesulfonic acid A. Stubna E. Mu¨nck M. P. Hendrich IM Inner membrane Department of Chemistry, Carnegie Mellon University, IMS Intermembrane space Pittsburgh, PA 15213-2683, USA NHE Normal hydrogen electrode OM Outer membrane P. A. Lindahl SH buffer 0.6 M sorbitol/20 mM N-(2-hydroxyethyl) Department of Biochemistry and Biophysics, 0 Texas A&M University, piperazine-N -ethanesulfonic acid buffer pH College Station, TX 77843, USA 7.4 123 1030 J Biol Inorg Chem (2007) 12:1029–1053

SP buffer 1.2 M sorbitol/20 mM potassium Another group of proteins are involved in iron trafﬁck- 2+ phosphate buffer pH 7.4 ing. Isa2p [38] and Yfh1p [39] help import Fe 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 Introduction synthase (Cox10p) and heme A synthase (Cox15p) bind intermediate states of hemes [33, 35, 45]. Cytochrome c

Mitochondria are the cellular organelles in which oxidative heme lyase (Cyc3p) and cytochrome c1 heme lyase (Cyt2p) phosphorylation and a myriad of related processes install heme c into cytochromes c and c1, respectively [46]. involving iron, copper and manganese occur. These bran- Mdl1p exports heme groups [47], while Atm1p and Erv1p ched tubular structures have an outer membrane (OM), an export Fe/S clusters [48, 49]. Coq7p is a yeast mitochon- aqueous intermembrane space (IMS), an inner membrane drial protein that contains a diiron center and serves as a (IM) and an aqueous matrix region. The IM is highly monooxygenase/hydroxylase in coenzyme Q (CoQ) bio- invaginated, with cristae protruding into the aqueous synthesis [50, 51]. matrix region. Imported iron ions are used in heme and Cytochrome c oxidase is the best-known copper-con- iron–sulfur (Fe/S) cluster biosynthesis. A portion of these taining mitochondrial protein. The Cox1p subunit of this nascent prosthetic groups are incorporated into mitochon- complex contains one copper ion (CuB) adjacent to heme drial apoproteins, while the remainder are exported to the a3 in its active site, while the electron-transfer CuA site in cytosol. Imported copper and manganese ions are installed Cox2p contains two copper ions [52]. Cox23p, Cox17p, into cytochrome c oxidase and manganese superoxide Sco1p and Cox11p are chaperones that import Cu ions into dismutase, respectively. mitochondria and insert them into Cox1p and Cox2p dur- The proteins involved in these processes can be cate- ing their assembly [53–55]. Copper ions in these chaper- gorized in terms of the metal centers they contain. Proteins ones are in the diamagnetic Cu+ oxidation state. A small containing Fe2S2,Fe3S4 and/or Fe4S4 clusters include amount of the cytosolic copper-containing (Cu–Zn) succinate dehydrogenase [1–3], the Rieske protein [4], superoxide dismutase (Sod1p) appears to localize in the aconitase and homoaconitase [5, 6], ferredoxin/adreno- IMS of mitochondria [56]. Approximately 90% of mito- doxin [7–10], biotin synthase [11–15] and lipoic acid chondrial copper is found in the matrix as a nonproteina- synthase [16–18]. Dihydroxyacid dehydratase catalyzes the ceously bound pool of Cu+ ions [57]. dehydration of an intermediate in the biosynthesis pathway Manganese superoxide dismutase (Sod2p) appears to be of branched-chain amino acids [19]. Although the metal the only manganese-containing enzyme in Saccharomyces center in this enzyme has not been well studied, the cerevisiae mitochondria. The mitochondrial manganese homologous enzyme from Escherichia coli contains an chaperone protein (Mtm1p) helps to import manganese Fe4S4 cluster [20]. Such clusters are also found in scaffold ions and to install one of these ions into matrix-localized proteins which are used in the synthesis of Fe/S clusters, apo-Sod2p [58]. including Isu1p, Isu2p, Isa1p and Nfu1p [21–24]. A Flavins and ubiquinone can be stabilized in an S = 1/2 BLAST search suggests that the open reading frame semiquinone state that affords electron paramagnetic res- YOR356W encodes the flavin adenine dinucleotide-con- onance (EPR) signals at the free-electron g value, 2.00. taining and Fe4S4-containing electron transfer flavoprotein Flavin-containing mitochondrial proteins include a-keto- (ETF) dehydrogenase [25–27]. glutarate dehydrogenase [59], D-lactate cytochrome c ox- Other mitochondrial proteins contain heme groups. idoreductases [60], glutathione reductase [61], thioredoxin Heme b is found in cytochrome bc1 [28], cytochrome c reductase [62], glycerol-3-phosphate dehydrogenase [63], peroxidase [29], succinate dehydrogenase and flavocyto- D-arabinono-1,4-lactone oxidase [64], acetolactate synthase chrome b2 [30]. Heme a is found in cytochrome c oxi- [65], methylene tetrahydrofolate reductase [66], succinate dase [31], while heme c is contained in cytochrome c1 dehydrogenase [67], Coq6p [68], Mmf1p [69] and ETF and in both isoforms of cytochrome c [32]. Heme dehydrogenase [25–27]. monooxygenase catalyzes the conversion of heme b to The most important spectroscopic technique that has heme a within the heme biosynthetic pathway [33]. The been applied to intact mitochondria is EPR, dating from the homolog from E. coli contains heme b and heme a pioneering work of Beinert [70], who initially described prosthetic groups [34–36]. When yeast cells are grown high-spin heme signals from cytochrome c oxidase [71]. + under respiratory conditions, the heme-b-containing cat- The gave = 2.01 EPR signal from the inactivated [Fe3S4] alase A (Cta1p) is targeted to the mitochondrial matrix form of aconitase was observed in crude intact rat-heart

[37]. mitochondria exposed to H2O2 [72]. EPR signals from the 123 J Biol Inorg Chem (2007) 12:1029–1053 1031

+ Rieske cluster of cytochrome bc1 and the [Fe2S2] cluster 25-L solution] in a custom-built thermostatically controlled of succinate dehydrogenase have also been observed in autoclavable 25-L glass fermenter in which cultures were intact mitochondria [73–78]. EPR spectra of intact mito- stirred and bubbled with pure O2 at a rate of approximately chondria were examined to determine the effect of abol- 3 L/min, dispersed through a fine glass frit with a diameter ishing heme biosynthesis on succinate dehydrogenase and of 5 cm. Under these growth conditions, cells ferment on the Rieske protein [75] and to determine the effects of Ca2+ glucose at early stages of growth and then switch to res- and Mn2+ ions [79, 80]. Adrenodoxin levels in intact piration on lactate once the glucose has been consumed. human placental mitochondria were examined by EPR Harvesting commenced when the optical density of a 1-cm [81]. Respiratory complexes in submitochondrial fractions solution at 600 nm reached 1.2–1.4. The culture was have also been examined [82–84]. In contrast, there has chilled to 278 K and harvested at 5,000 rpm using a been just one report of a Mo¨ssbauer spectrum of intact Sorvall SLC-6000 rotor and a Sorvall Evolution centrifuge. mitochondria, specifically of a strain in which yfh1 was Immediately after harvesting and without freezing the deleted [39]. This genetic modification causes iron to pelleted cells, mitochondria were isolated essentially as accumulate in the matrix, and the observed Mo¨ssbauer described in [85] except that all steps were performed spectral intensity exclusively reflected the accumulated anaerobically. Cell paste (100–150 g) was transferred into iron. The ‘‘control’’ Mo¨ssbauer spectrum of wild-type a refrigerated argon-atmosphere glove box (M. Braun) mitochondria was devoid of any signals. maintained at approximately 278 K and approximately

This overview highlights the complexity of transition 1 ppm O2 as monitored continuously using a model 310 metal metabolism occurring within these organelles. We Teledyne analyzer. Buffers used in the isolation were report on our efforts to establish a few simple yet unes- degassed on a Schlenk line. For some preparations, all tablished aspects of iron metabolism in yeast mitochondria, isolation buffers were supplemented with ethylenedi- namely, the absolute concentration of iron and of overall aminetetraacetic acid (EDTA) or ethylenebis(oxyethy- protein contained therein, and the proportion of that iron lenenitrilo)tetraacetic acid (EGTA) (Acros) at final present in various types of centers (e.g., hemes, Fe/S concentrations of 1 or 10 mM. In other preparations, no clusters, etc.). Our approach was to investigate mitochon- metal chelators were included. Cell paste was suspended dria from S. cerevisiae using EPR and Mo¨ssbauer spec- in a 100 mM tris(hydroxymethyl)aminomethane sulfate/ troscopy along with various bioanalytical characterizations. 10 mM dithiothreitol (DTT) buffer (500 mL) and then For the first time using whole mitochondria, the absolute spun at 5,000 rpm for 5 min in the SLC-6000 rotor. Sub- spin concentrations of detectable metal protein species sequent centrifugations were performed under these con- have been quantified from EPR spectra. We investigated ditions unless otherwise stated. The resulting pellet was intact mitochondria prepared under different redox and/or suspended in 1.2 M sorbitol/20 mM potassium phosphate isolation conditions. We determined the proportion of buffer, pH 7.4 (500 mL), hereafter referred to as SP buffer, excluded buffer in these packed samples, which, when using a rubber policeman. The resulting suspension was combined with metal and protein determinations of the centrifuged, resuspended in SP buffer (500 mL), centri- packed samples, allowed us to estimate the absolute iron fuged again, and resuspended again in the same buffer. Cell concentration contained within these organelles. This walls were disrupted by adding 3 mg of 100 units/mg yeast information, when combined with our spectroscopic lytic enzyme (Sigma) per gram of cell paste. The resulting results, allowed us to estimate, albeit in broad terms, how spheroplasts were centrifuged, suspended in SP buffer iron is distributed within the organelle. This distribution (500 mL) and then centrifuged. The pellet was resuspended was then compared with that calculated from the iron- in 250 mL of 1.2 M sorbitol/40 mM N-(2-hydroxyethyl) containing proteins known to be present in the mitochon- piperazine-N0-ethanesulfonic acid (HEPES) pH 7.4 and drial proteome. 250 mL of 1 mM phenylmethylsulfonyl fluoride in double-

distilled H2O. The mixture was homogenized using 25 strokes of a 40-mL Dounce homogenizer (Fisher Scientific) Materials and methods during a period of 2–4 min. The suspension was centrifuged at 2,500 rpm for 5 min, and the supernatant was Cell growth and isolation of mitochrondia transferred to a fresh centrifuge bottle and centrifuged again under the same conditions. The supernatant, which S. cerevisiae cells (strain D273-10B) were grown on SSlac consisted of crude mitochondria, was then centrifuged at medium (0.3% glucose, 1.7% lactate) [72 g yeast extract, 10,000 rpm in a Sorvall SLA-1500 rotor for 10 min. The 25 g ammonium chloride, 25 g potassium hydrogen phos- resulting pellet was resuspended in 200 mL of a 0.6 M phate, 12.5 g NaCl, 12.5 g CaCl2, 14.4 g MgCl2, 12.5 g sorbitol/20 mM HEPES buffer pH 7.4, hereafter referred to glucose and 0.7 L of 60% sodium lacate syrup (Fisher) in as SH buffer. The resulting solution was centrifuged three 123 1032 J Biol Inorg Chem (2007) 12:1029–1053 more times, in the manner described in the previous three potassium ferrocyanide (w/v) in SH buffer. This was fol- sentences, and the final pellet of crude mitochondria was lowed by en bloc staining using 1% uranyl acetate in SH resuspended in 20 mL SH buffer. This solution was loaded buffer. Samples were dehydrated by incubation in onto a discontinuous gradient solution composed of 10 mL increasingly concentrated ethanol solutions and then of 15% and 10 mL of 20% (w/v) Histodenz1 (Sigma) embedded using epoxy-based resin. Thin-sectioning was prepared in SH buffer and contained in Beckman Ultra performed using a glass knife/water trough on a micro- ClearTM centrifuge tubes. The tubes were placed in the tome, followed by retrieval of the thin sections using 200 buckets of an SW-32Ti rotor (Beckman Coulter). The mesh grids. Positive staining of these sections was per- buckets were sealed, removed from the box and spun at formed using lead acetate/sodium hydroxide [88]. Images 9,000 rpm in an SW-32Ti rotor (Beckman Coulter) for were obtained using a JEOL 1200 EX transmission electron 1.5 h using a Beckman L7 ultracentrifuge. The buckets microscope. were returned to the box, and the tubes were placed in a For fluorescence images, equivalent mitochondrial solu- support which allowed the pure mitochondrial band at the tions were incubated in SH buffer, containing 500 nM interface of the gradient to be collected after first removing MitoTracker1 (Molecular Probes) or, in another experi- the layer above the band. From 150 g cell paste, a total of ment, 1 lM ERTracker1 at 310 K for 45 min. The solution 5–15 mL of mitochondrial solution in the ‘‘as-isolated’’ was centrifuged, and the pellet was resuspended in SH buf- state was obtained using three to six buckets depending on fer. Images were obtained using a Bio-Rad Radiance the yield. The only reductant used during the procedure 2000 MP instrument equipped with a ·63 (water-immer- was DTT and then only at an early step of the isolation sion) objective. procedure before cell walls were disrupted. E0 for the disulfide/DTT half-cell is 330 mV versus the normal hydrogen electrode (NHE) [86]. Anaerobically prepared Oxygen consumption measurements isolation buffers undoubtedly contained a trace of oxidizing ability [87]. Both factors considered, the resulting A sample of non-chelator-treated intact mitochondria was solution potential of mitochondria in the non-redox-buf- suspended in SH buffer. A 5-mL portion was assayed for fered ‘‘as-isolated’’ state was estimated to be between 0.1 protein concentration using the biuret method [89]as and 0 mV versus NHE. Prior to freezing, some samples described in ‘‘Protein and metal ion concentrations.’’ were exposed to air (typically for 1 day at 277 K), sodium Another portion of the intact mitochondrial solution was dithionite (10 mM at pH 7.5 or 8.5), potassium ferricyanide divided into three samples. One sample was incubated (1 mM) or nitric oxide (1 atm). anaerobically for 4–5 h with 10 mM EDTA, another was For Mo¨ssbauer spectroscopy studies, S. cerevisiae cells incubated similarly with 10 mM EGTA, and the remaining were grown similarly except that the medium was sup- sample was not treated. Each sample (1.2 mL) was injected 57 plemented with 20 lM FeCl3. With use of a custom- into 29 ± 1 mL of air-saturated SH buffer containing TM made Delrin insert that fit in the buckets of the SW-32Ti 1.5 mM NADH, 0.2 mM ADP, 2 mM MgCl2,20mM rotor, isolated mitochondria were packed tightly into phosphates pH 7.4, 250 mM sucrose and 10 mM KCl, Mo¨ssbauer cuvettes by centrifugation, typically at essentially as described in [90]. The solution was main- 9,000 rpm for 2 h. Samples were then frozen inside the tained at 298 K in a water-jacketed glass vessel which glove box by contact with a liquid-nitrogen-cooled alumi- contained no gas head space. Included in this vessel was a num block. There was some variation in speed and duration Clark oxygen electrode (YSI Bioanalytical Products). The used in packing, resulting in some differences in terms of final protein concentration was 0.10 mg/mL. observed 57Fe concentrations. Each spectrum presented here was recorded with an approximately 40 mCi 57Co source. Electron paramagnetic resonance

Custom-built DelrinTM inserts were designed to fit within Electron and fluorescence microscopy the buckets of the SW-32Ti rotor. Holes were drilled into the center of these inserts, with a diameter just sufficient to One milliliter of as-isolated mitochondrial solution was fit a modified EPR tube (4.96-mm outer diameter; 3.39-mm microcentrifuged (Fisher Scientific) at 6,400 rpm for 5 min inner diameter; 80-mm long; Wilmad/Lab Glass, Buena, in a 1.5-mL Eppendorf tube. The pellet was resuspended in NJ, USA). A 2-mm-long cylinder of silicone rubber was SH buffer and glutaraldehyde (2.0% v/v final concentra- inserted at the bottom of the hole. The brown mitochon- tion) was added. The solution was recentrifuged and the drial solution obtained from the gradient step described in pellet was resuspended in 1% osmium tetroxide and 0.5% ‘‘Cell growth and isolation of mitochondria’’ was diluted 123 J Biol Inorg Chem (2007) 12:1029–1053 1033 with an equal volume of SH buffer. Tubes were filled with colorimetric method. Relative to amino acid analysis, the this solution and the entire assembly was sealed, removed results obtained using the biuret method were similar from the box and spun by centrifugation at 9,000 rpm for within the uncertainty of the measurements. For quantita- 1 h. Samples were returned to the box, and the supernatant tive amino acid analysis, aliquots were hydrolyzed in 6 M was replaced with additional mitochondrial solution. This HCl/2% phenol at 383 K and analyzed using a Hewlett- process was repeated until the volume of tightly packed Packard AminoQuant system. Amino acid percentages mitochondria at the bottom of the tube reached approxi- were similar among preparations. Primary and secondary mately 400 lL. EPR tubes were removed from the inserts amino acids present in the samples were derivatized and frozen in less than 1 min using liquid N2. Two to four using o-phthalaldehyde and 9-fluoromethylchloroformate, EPR samples were prepared from a solution of gradient- respectively. Metal concentrations were determined by purified mitochondria isolated from 25 L of culture. atomic absorption spectrometry (PE AAnalyst 700 oper- One end of a stainless steel wire (20 cm · 0.5-mm ating in furnace mode) and by inductively coupled plasma diameter) was attached to one end of a stainless steel rod mass spectrometry (PerkinElmer). Sonicated samples (20 cm · 4.8-mm diameter), with the wire extended (250–400 lL) were digested using an equal volume of coaxially with the rod. Approximately 5 cm beyond the 15.8 M trace-metal-grade HNO3 (Fischer Scientific) in a point of attachment, the wire was bent back towards the rod sealed plastic tube that was then incubated overnight at (like a hairpin) and coiled around itself up towards the rod. 353 K. The resulting solution was diluted with deionized

The outer diameter of the coil at the base of the hairpin was and distilled H2O to a final HNO3 concentration of 0.2 M. 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 Percentage of external solution in packed samples tube. In this way, the wire coil fit snugly into the upper region of the EPR tubes. The entire assembly was just Custom-built Lexan ‘‘graduated cylinders’’ were con- sufficiently robust to be inserted into and removed from the structed within inserts that fit within buckets of the SW- EPR cavity. Spectra were obtained with a Bruker EMX X- 32Ti rotor (Fig. 1). These inserts were used to accurately band EPR spectrometer operating in perpendicular mode measure the volume of a packed mitochondria sample, with an Oxford Instruments EM910 cryostat. Signals were obtained by loading a solution of isolated mitochondria and simulated with SpinCount written by one of the authors spinning the sample for 1 h at 9,000 rpm (10,000g). The (M.P.H.). Signal intensities were quantified relative to a supernatant was decanted and the volume of the packed CuEDTA spin standard using the same software. sample was measured using this apparatus. This volume

(Vpel) was assumed to be composed of the volume of the mitochondria plus the volume of excluded water: 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 Fig. 1 Graduated cylinder used to measure the volume of packed is the most accurate method available [91], or by the biuret mitochondria samples 123 1034 J Biol Inorg Chem (2007) 12:1029–1053

Table 1 Determination of excluded buffer in packed mitochondria samples

* * * Cstock Vstock Csup1 Vsup1 VH2O1 Csup2 Vsup2 VH2O2 Vpel Average (cpm/mL) (mL) (cpm/mL) (mL) (mL) (cpm/mL) (mL) (mL) (mL) %H2OInVpel

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

(Vpel = Vmito + VH2O). To determine the ratio VH2O/Vpel,a while others were isolated in the presence of either EDTA 1.00-mL stock solution of radioactively labeled sucrose or EGTA. These chelators were added to remove adven- (American Radiolabeled Chemicals, 625 mCi/mmol), pre- titious metal ions associated with mitochondria. EGTA is * pared in SH buffer (with Cstock in counts per minute per unable to penetrate biological membranes [92], while this milliliter given in Table 1 for each experiment), was added property is uncertain with respect to EDTA. However, to the pellet and the pellet was resuspended. The inserts EDTA has been used in isolating mitochondria [93] and as were spun by centrifugation as described above, the far as we are aware, there have been no reports of EDTA supernatant was removed, the volume (Vsup1) was deter- stripping essential metal ions from these organelles. mined using a gastight syringe (Hamilton), and the con- We assayed a number of preparations for purity and * centration of radioactivity (Cstock) was determined by membrane integrity using electron microscopy. Although scintillation counting (Beckman 5000SL). Assuming that significant size dispersion was typically evident (Fig. 2, none of the sucrose entered the mitochondria, the excluded top), there was no obvious evidence of impurities (bacteria water will also have a concentration of radioactivity given or Golgi apparatus) or disrupted membrane structures. * by Cstock. The conservation of matter suggests that Sample morphology was independent of the method of isolation (as-isolated, EDTA-treated or EGTA-treated). CstockVstock ¼ Csup1Vsup1 þ Csup1VH2O1 : Our images are similar to those obtained in the classical studies of Hackenbrock [94] and more recently [95]. Dis- This equation was solved for V . The resulting pellet H2O1 persion probably results from the dynamic fission and was found to have essentially the same volume as the fusion processes that occur in yeast mitochondria [96]. original pellet. This pellet, containing radioactively labeled Confocal microscopic images reveal that mitochondria sucrose in the external volume, was resuspended with a form extensive tubelike networks extending throughout the solution of nonradioactively labeled sucrose, and the other cell [97]. These dynamic changes in size and shape would steps of the same process were repeated. In this case, the appear to render the concept of the number of mitochondria resulting concentration of radioactivity in the supernatant per cell rather meaningless. A more quantifiable parameter fraction was called C* and the corresponding sup2 is the volume occupied by these organelles, and we will use conservation of matter relationship becomes this parameter throughout this paper. Fluorescence microscopy was also used to assess purity. Csup1 VH2O2 ¼ Csup2 Vsup2 þ Csup2 VH2O2 : One sample was stained for fluorescence with MitoTrac- 1 1 This equation was solved for VH2O2 . The average of the two ker , while another was stained with ERTracker . The values for VH2O was divided by Vpel, affording the fraction former dye associates with mitochondria, while the latter of the pellet volume due to excluded water. associates with the endoplasmic reticulum. As shown in Fig. 2, the vast majority of objects in our samples assimi- lated the MitoTracker1 stain. There was no obvious sign of Results endoplasmic reticulum contamination using ERTracker1 (data not shown). Both results suggest that the samples Characterization of intact mitochondria examined here were relatively pure and intact. We assayed a number of preparations for their ability to

Intact yeast mitochondria were isolated as described in consume O2. As shown in Fig. 3, preparations incubated in ‘‘Materials and methods.’’ Some preparations were isolated the absence of chelator or in the presence of EDTA or EGTA without adding a metal chelator to the isolation buffers, consumed 240, 160 and 200 nmol O2 per minute per 123 J Biol Inorg Chem (2007) 12:1029–1053 1035

300

250

200 n

e 150 g y x O

M 100 µ

0 2 4 6 8 101214161820 Time (min)

Fig. 3 Oxygen consumption by isolated intact mitochondria. No chelator (squares), ethylenediaminetetraacetic acid (EDTA; trian- gles), ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA; circles). The experiment was performed as described in ‘‘Materials and methods’’

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 lMFe(n = 11), 220 ± 150 lMCu(n = 11), Fig. 2 Electron microscopy (top) and fluorescence microscopy 20 ± 10 lMMn(n = 11) and 330 ± 170 lMZn(n = 5). (bottom) images of whole mitochondria isolated from Saccharomyces cerevisiae 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 milligram of protein, respectively (estimated relative error the protein and Fe concentrations measured in samples of ±20%) when incubated in buffer containing NADH. isolated in the presence of chelators indicates that these Control samples assayed in the absence of NADH consumed concentrations (and their ratio, approximately 10 nmol little O2. These rates are similar to those reported previously Fe/mg protein) are reliable within a relative uncertainty of [98–100]. We also evaluated the coupling ratio of our 25%. preparations, defined as the rate of O2 consumption with Next, we determined the proportion of the packed ADP added to the assay solution divided by the rate of mitochondria samples due to the mitochondria themselves consumption when ADP was absent. In our fresh samples, (rather than to excluded solution). Using the procedure this ratio was approximately 2, similar to previously reported described in ‘‘Materials and methods,’’ we found the per- ratios [99, 100], whereas it approached 1 for mitochondria centage of mitochondria in our packed samples to be stored anaerobically at 278 K for approximately 3 days. 77 ± 7 (n = 7), as shown in Table 1. The absolute con- Preparations used for EPR and Mo¨ssbauer analyses were centrations of protein and Fe concentrations contained in frozen between 6 h and 3 days after they were isolated. We ‘‘neat’’ mitochondria (devoid of solvent) could then be have not yet been able to correlate the age of the mito- calculated by dividing the measured concentrations for the chondria to specific spectral changes, but we suspect that packed samples by 0.77, affording a protein concentration spectral features might become slightly broader with age. of approximately 70 mg/mL and Fe concentrations of 0.74 We determined protein and metal concentrations in our and 1.1 mM for samples isolated in the presence and packed samples. The mean protein concentration (n = 15) absence of chelators, respectively. Given the uncertainty as

123 1036 J Biol Inorg Chem (2007) 12:1029–1053 to whether the Fe removed by chelators had any functional region (Fig. 5, spectrum A) is often dominated by a signal relevance, and on the basis of 57Fe concentration estimates with a six-line hyperfine pattern (magnetic hyperfine cou- based on Mo¨ssbauer intensities (see ‘‘Mo¨ssbauer spectra of pling constant, a = 90 G) typical of an S = 5/2 Mn(II) mitochondria’’), we conclude that the concentration of Fe species. A feature at g * 1.94 is also evident but is in respiring yeast mitochondria is 800 ± 200 lM. 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 EPR of mitochondria absence of chelators, exhibited a g = 4.3 signal with significantly lower intensities than that in Fig. 4, spectrum A Mitochondria prepared in three different redox states, and did not show the Mn(II) signal of Fig. 5, spectrum A; including as-isolated, oxidized and reduced, were packed rather it exhibited the spectrum shown in Fig. 5, spec- tightly into custom-designed EPR tubes so as to expel trum B (discussed later). The g & 4.3 and Mn(II) signals external buffer and maximize the intensity of mitochon- were also either absent or present at low intensity in spectra drial EPR signals. As-isolated samples are defined as those of samples as-isolated with chelators included in all iso- prepared anerobically in the absence of either oxidant or lation buffers. This suggests that all or most of the species yielding these signals arise from adventitious Mn2+ and reductant. Oxidized samples were treated with either O2 or 3+ ferricyanide. Reduced samples were treated with sodium Fe ions that can be chelated by EDTA and EGTA. 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 g an attempt to distinguish EPR signals originating from 87 6 5 4 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 6.9 5.0 and reanalyzed spectroscopically). Once thawed, samples were used for protein and metal analyses and then dis- A carded. This procedure produced reasonable but not perfect 6.4 correlation between the redox state in which the sample was prepared and the types and intensities of EPR signals 5.4 observed. B EPR signals observed during this study are shown in 6.0 Figs. 4 and 5. The principal g values for these signals and associated spin concentrations are compiled in Table 2. C EPR signals of high-spin (S = 5/2) Fe3+ species were analyzed with the conventional spin Hamiltonian D 2 2 2 H ¼ D½S 35=12 þ E=DðS S Þ þ g0bS B: z x y E

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 80 100 120 140 160 180 200

E/D and the intrinsic g value g0 & 2.0 [101]. B (mT) Mitochondria prepared in the absence of metal chelators sometimes exhibited a signal at g = 4.3, as shown in Fig. 4, Fig. 4 Low-ﬁeld X-band electron paramagnetic resonance (EPR) spectra of intact mitochondria. A Non-chelator-treated, as-isolated, B spectrum A. This signal is typical of non-heme Fe3+ spe- EGTA-treated, O2-exposed, C EGTA-treated, as-isolated, D EGTA- cies with E/D & 0.27–0.33. Such spectra also typically treated, reduced with 10 mM dithionite pH 7.4, E same as D but at pH include features at g = 6.9 and 5.0, indicative of a high- 8.5. EPR conditions as follows: average microwave frequency, 3+ 9.45 GHz; microwave power, 20 mW; modulation amplitude, 10 G; spin Fe heme with E/D = 0.041. The minor signal at 4 4 3+ receiver gain 1 · 10 for A–C, 5.02 · 10 for D and E. Temperature, g = 6.0 indicates a second high-spin Fe heme with 10 K. The intensities of D and E have been multiplied by 5 and 2, E/D & 0 (discussed later). In such samples, the g =2 respectively

123 J Biol Inorg Chem (2007) 12:1029–1053 1037

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 O2 prior to centrifugation and freezing under standard anaerobic conditions. In chelator- treated 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 (gave = 1.94), as in Fig. 5, spectrum B. The microwave power which caused the gave = 1.94 signal intensity divided by the square root of the power to reach

half maximum was P1/2 = 57 mW at 10 K. On closer inspection, a second signal, with g = 2.02, 1.90 and 1.75

(gave = 1.90) is also evident. The g values of the gave = 1.94 and 1.90 signals strongly suggest that they arise from Fe/S proteins.

An isotropic giso = 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 Fig. 5 High-ﬁeld X-band EPR spectra of intact mitochondria. A Non- the sharp giso = 2.00 signal is easily saturated at less than chelator-treated, as-isolated, B a more recent preparation of non- 1 lW. The other signals in the spectrum begin to saturate chelator-treated, as-isolated (200 lW), C EDTA treated, NO-exposed 4 (9.458 GHz, 200 lW, gain 5.02 · 10 ), D EGTA-treated, O2- at powers greater than 80 lW. exposed, E EGTA-treated, oxidized with 1 mM ferricyanide. Other A fourth signal with one principal g value near 2.08 can conditions were as for Fig. 4 except that the average microwave also be observed in many preparations; however, the other frequency was 9.43 GHz. The intensities of B–E have been multiplied associated g values are poorly resolved at 10 K. Spectral by 5, 5, 2, and 5, respectively. Microwave power in A, D, and E was 200 lW overlap became less problematic at 130 K, as this signal remains slow-relaxing, while the gave = 1.94 and 1.90 sig- The same two high-spin heme species described above nals are broadened at that temperature; spectra collected at were also observed in spectra of chelator-treated as-iso- that temperature suggest that the other features associated lated preparations, but an additional high-spin heme signal 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 Concentration Tentative assignment (g1, g2, g3) range (lM)

High-spin Fe3+ heme 1 S = 5/2, E/D = 0.041 6.9, 5.0 0–3 Cytochrome c peroxidase (Ccp1p) 3+ High-spin Fe heme 2 S = 5/2, E/D = 0.021 6.4, 5.4 0–2 Cytochrome c oxidase, heme a3:Cub 3+ High-spin Fe heme 3 S = 5/2, E/D = 0 6.0 0–1 Cytochrome c oxidase, heme a3:Cub g = 4.3 S = 5/2, E/D = 0.33 4.27 Minor to 40 Adventitious Fe3+ gave = 2.02 S = 1/2 or spin-coupled 2.085, 1.989, 1.985 1–20 Unassigned; possibly spin-interacting system Fe/S clusters of ETF dehydrogenase + gave = 2.01 S = 1/2 2.026, 2.022, 2.003 0–5 [Fe3S4] 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 Mn2+ g = 2.00 (isotropic) S = 1/2 2.000, 2.000, 2.000 <2 C- or O-based organic radical + gave = 1.94 S = 1/2 2.026, 1.934, 1.913 0–23 Succinate dehydrogenase [Fe2S2] (Sdh2p) + gave = 1.90 S = 1/2 2.025, 1.897, 1.784 0–45 Rieske [Fe2S2] cluster (Rip1p) ETF electron transfer ﬂavoprotein

123 1038 J Biol Inorg Chem (2007) 12:1029–1053

gave = 2.02 for the signal. This was conﬁrmed by spectral spectrum C). This signal is characteristic of a pentacoor- simulation and decomposition. The 10 K and 200 lW dinate heme–nitrosyl complex [101]. The spin concentra- spectrum of an EGTA-treated sample was decomposed tion associated with this signal (20 lM) was quite high,

(Fig. 6, spectrum A, solid line) by simulating the gave = and it may reflect the overall concentration of pentacoor- 1.90 (Fig. 6, spectrum B), 1.94 (Fig. 6, spectrum C), 2.00 dinate Fe2+ heme species present, as such species are (Fig. 6, spectrum D) and 2.02 (Fig. 6, spectrum E) signals, known to bind to NO to yield similar 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 Mo¨ssbauer spectra of mitochondria the experimental spectrum. Experimental spectra from other preparations gave similar deconvolutions. For readers not familiar with details of Mo¨ssbauer spectroscopy we have given a short tutorial-type section in the supplementary material. For the present study we have EPR of intact mitochondria treated with various collected Mo¨ssbauer spectra from numerous samples of redox agents intact mitochondria. A spectrum from an as-isolated sample not exposed to metal chelators, shown in Fig. 7, spec- Earlier mitochondrial preparations that had been exposed trum A, exhibits three distinct spectral features (similar to air for 1–2 days exhibited low-field regions essentially spectra were observed for preparations treated with metal devoid of heme-containing signals. More recent prepara- chelators, EGTA and EDTA). Approximately 15–20% of tions, exposed to O2 for 6 h, exhibited the high-spin heme the iron belongs to a doublet with quadrupole splitting signal at g = (6.4, 5.4) at high concentration (Fig. 4, DEQ & 3.3 mm/s and isomer shift d & 1.3 mm/s; this spectrum B). In these samples, the g = 2 region generally doublet is outlined in the experimental spectrum. The 2+ consisted of intense signals with gave = 2.01 and giso = quoted values are typical of high-spin mononuclear Fe 2.00, and were largely devoid of gave = 1.94 and 1.90 ions in penta- or hexacoordinate nitrogen/oxygen envi- II signals (e.g., Fig. 5, spectrum D). A similar set of signals ronments: Fe (H2O)6 complexes, the iron sites in reduced were observed in a sample oxidized with ferricyanide. In dioxygenases and iron superoxide dismutase, and of fully this case, the low-field region displayed a mixture of the reduced binuclear iron-oxo centers at low applied field. g = 6.0 and g = (6.4, 5.3) high-spin heme signals, while High-spin Fe2+ hemes have distinctly smaller d values the high-field region revealed an intense gave = 2.01 signal (approximately 0.83–0.93 mm/s); however, such species (spin concentration approximately 5 lM) (Fig. 5, spec- would be difficult to resolve if they were to account for less trum E) along with a sizable giso = 2.00 signal (1 lM) and than 5% of the Fe in the present samples. low-intensity gave = 1.94 and 1.90 signals. A second doublet in Fig. 7, spectrum A (outlined as the The gave = 2.01 signal was not observed in spectra of dashed line in Fig. 7, spectrum B), accounting for 55–65% 1 samples treated with dithionite or in spectra of most as- of the iron, has DEQ & 1.15 mm/s and d & 0.46 mm/s. isolated preparations, indicating that the species exhibiting This doublet most probably represents Fe4S4 clusters in the 2+ this signal is EPR-silent when reduced. The low-field 2+ core oxidation state. In this state [Fe4S4] clusters have region of spectra from samples treated with dithionite was a ground state with cluster spin S = 0. Low-spin Fe2+ he- largely devoid of signals, as expected from the thermody- mes such as cytochromes b and c have very similar DEQ namic ability of dithionite to reduce Fe3+ hemes. The and d values and thus their contributions would be difficult 2+ gave = 1.94 and 1.90 signals were present, as expected, but to separate from those of [Fe4S4] clusters. In principle, with concentrations similar to that observed in as-isolated the cytochromes should be oxidizable and thus detectable samples. The reduction ability of dithionite declines as pH by EPR; however, no such signals were identified in the is lowered [102], and we anticipated that the intensity of analogous samples examined by EPR. Thus, we suspect these signals might increase significantly at pH 8.5 relative that low-spin Fe2+ hemes do not contribute substantially to to the intensity at pH 7.4. This expectation was not ful- 1 4 filled. However, an unresolved absorption-like feature at For a purified Fe S4 ferredoxin the area under the doublet can be g * 6.4 was observed in spectra of a sample reduced with quantified to within 1–2%. Here, the uncertainties are considerably larger, primarily because more than one cluster contributes. The dithionite at pH 8.5 (Fig. 4, spectrum E) but not at pH 7.4 primary contributors to the doublet may be aconitase and dihydroxy- (Fig. 4, spectrum D). This may be associated with S = 3/2 acid dehydratase. Because species with slightly different but + unresolved parameters contribute, lineshapes are heterogeneously [Fe4S4] clusters [103]. Another preparation was exposed to 1 atm NO. This broadened Lorentzians. We used both the Lorentzian and the Voight lineshape options of WMOSS. As Voight shapes are narrower at the afforded a signal at g\ = 2.07 and a g|| = 2.01 resonance base, this option yields, upon visual inspection, a lower estimate for that exhibited a 14N hyperfine splitting of a = 14 G (Fig. 5, the concentration. 123 J Biol Inorg Chem (2007) 12:1029–1053 1039

0 A

0.0 Absorption (%)

C 0.5 8 T

-5 0 5 Velocity (mm/s)

Fig. 7 Low-temperature (4.2 K) Mo¨ssbauer spectra of intact as- isolated mitochondria. A Spectrum of a preparation not treated with chelators, recorded in a 45 mT applied magnetic field. The spectrum of high-spin Fe2+ components is outlined above the experimental data. B EGTA-treated mitochondria with magnetic field as for A. The dashed line indicates the DEQ = 1.15 mm/s doublet, a component 2+ comprising predominantly [Fe4S4] clusters. The solid line repre- 2+ 2+ sents the sum of the Fe and [Fe4S4] 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 DEQ = 1.15 mm/s component is diamagnetic. The solid line 3 Fig. 6 above the data is a simulation for a high-spin Fe component, and the Simulations of the EPR spectrum of Fig. 5, spectrum B for as- 3+ 2+ A solid line solid line drawn through the data is the sum of the Fe and [Fe4S4] isolated mitochondria without chelator. Data ( ) and sum 2+ (dashed line) of simulations (B–E). The g values of the simulations species. The Fe component was not simulated because this would require use of many unknown parameters. D The 45-mT spectrum of are stated for species with gave and concentrations of B 1.90, 44 lM; C l D l E l mitochondria treated with O2/antimycin. The solid line outlines the 1.94, 23 M; 2.00, 2 M; 2.02, 17 M. Experimental 2+ conditions are the same as for Fig. 5, spectrum B contributions of the DEQ = 1.15 mm/s doublet (52%) and the Fe component (12%) this doublet. We comment further on the DEQ = 1.15 mm/s component when we discuss the spectrum of Fig. 7, dioxygenases. Thus, with our equipment, a 5-mm-thick spectrum C. frozen aqueous solution sample containing 1 mM 57Fe A third component present in Fig. 7, spectrum A exhibiting a quadrupole doublet of 0.30 mm/s full width at exhibits broad absorption extending over a velocity range half maximum yields 5% resonance absorption. Using this of roughly 10 mm/s; this feature reflects unresolved mag- empirical rule (comparing the total absorption area to that netic hyperfine structure of (mostly) high-spin Fe3+ ions as under the ‘‘standard’’ doublet), the sample of Fig. 7, spec- well as other unidentified low-spin magnetic species. trum A has a 57Fe concentration of approximately 0.5 mM. Finally, as much as 12% of the total iron may belong to A similarly prepared sample (Fig. 7, spectrum B), but + 57 S = 1/2 [Fe4S4] clusters (discussed below). treated with the chelator EGTA, has an Fe concentration In principle, Mo¨ssbauer spectroscopy can be used, with of approximately 0.3 mM (both calculations taking into some effort and proper calibration, to determine the abso- effect the solvent void volume). Although unsupplemented lute 57Fe concentration of a sample. We have done this for with Fe, the media in which the yeast were grown certainly many years, mainly for keeping track of 57Fe enrichment in contained some natural-abundance Fe; thus, these Mo¨ss- proteins, which we have calibrated with ferredoxins and bauer spectroscopy-based estimates may be somewhat

123 1040 J Biol Inorg Chem (2007) 12:1029–1053

Table 3 Summary of Mo¨ssbauer and EPR results

Fe center O2/antimycin As-isolated, As-isolated, Dithionite-treated EPR no chelator EGTA

2+ 2+ S = 0 [Fe4S4] + low-spin Fe 52–57% 55–65% 40–50% 45% + S = 1/2, 3/2 [Fe4S4] <8% <12% 40% to minor 6% (gave = 2.02) 0/+ S = 2, 1/2 [Fe3S4] <5% <5% <5% <5% 3% (gave = 2.01) 2+ S = 0 [Fe2S2] <5% ND ND ND + S = 1/2 [Fe2S2] <12% <12% 10% High-spin Fe3+, octahedral, N/O ligands 5% ND 15% 1% + adventitious High-spin Fe2+ 5/6-CN, O/N ligands 12% 15–20% 20% 20% Low-spin Fe3+ ND ND EGTA ethylenebis(oxyethylenenitrilo)tetraacetic acid, ND not determined lower than those obtained by chemical analysis since spectrum A) and 8.0 T (Fig. 8, spectrum B). For this Mo¨ssbauer spectroscopy only detects 57Fe in our samples. sample approximately 45% of the 57Fe is found to be

The EGTA-treated sample (Fig. 7, spectrum B) contains associated with the DEQ = 1.15 mm/s doublet. Compared essentially the same spectral components as the sample that with the Fig. 7, spectrum A, there is increased absorption was not treated with chelators (Fig. 7, spectrum A); how- (from various paramagnetic species) around 1.8 and ever, the proportions were somewhat different, with 40– +2.2 mm/s Doppler velocity (arrows). These features most + 50% in the DEQ = 1.15 mm/s doublet, approximately 20% probably belong to S = 1/2 [Fe4S4] clusters. The solid high-spin Fe2+, 15% high-spin Fe3+ ions and some as yet lines overlapping the data in Fig. 8 are simulations typical 2+ unidentified iron. These percentages for the samples dis- of S = 0 [Fe4S4] clusters (drawn as the dashed lines to + cussed in this study are summarized in Table 3. Figure 7, represent 45% of total Fe) and from S = 1/2 [Fe4S4] spectrum C was recorded at 4.2 K in the presence of an (40%) clusters; the latter are, somewhat arbitrarily, repre- external magnetic field of 8.0 T applied parallel to the sented by two cluster forms using the parameters of c beam. The central feature, outlined separately by the reduced aconitase (20%) and a (generic) set of parameters 2+ + dashed line, belongs to the feature assigned to [Fe4S4] similar to those of the [Fe4S4] cluster E. coli sulfite clusters. This simulation was generated with the assump- reductase (20%). These two spectral components which + tion that the DEQ = 1.15 mm/s doublet represents iron have been used to represent the [Fe4S4] state could also be residing in a diamagnetic (S = 0) environment, in good drawn into Fig. 7, spectrum A, each representing 10% of agreement with the data. The two absorption bands at +8 the 57Fe in that spectrum. Interestingly, with the present and 8 mm/s Doppler velocity belong to high-spin Fe3+ decomposition, approximately 85% of the 57Fe would species with N/O octahedral coordination; a spectral sim- belong to Fe4S4 clusters in the dithionite-reduced sample. ulation (solid line) is shown above the data. This compo- This estimate is probably a bit high, as some of the 57 + nent, representing approximately 15% of the Fe, is absorption attributed to [Fe4S4] clusters may result from 3+ + probably a collection of various mononuclear Fe species [Fe2S2] clusters (but not more than 12%; see next para- 2+ with octahedral N/O ligation; such species typically have graph). Additional absorption attributed to [Fe4S4] clus- zero-field splitting parameters |D| < 2/cm and isotropic ters may also arise from low-spin Fe2+ cytochromes. In a magnetic hyperfine coupling constants A0 & (27–29) low external field, oxidized Fe3S4 clusters would be present MHz.2 as an S = 1/2 absorption extending beyond the central 2+ Figure 8 shows 4.2 K Mo¨ssbauer spectra of mitochon- [Fe4S4] doublet. While we see no direct evidence for the dria treated with 10 mM dithionite at pH 8.5; the spectra presence of such species, they could be present at con- were recorded in parallel applied fields of 50 mT (Fig. 8, centrations below 5% of the total 57Fe. We attempted to oxidize anaerobically isolated mito- 2 In weak applied fields, the lowest three Kramers doublets of the chondrial samples by exposing them to air for 1–2 days. spin sextet are generally populated at 4.2 K, yielding three Mo¨ssbauer Such treatment had little effect on Mo¨ssbauer spectral spectra per site. Moreover, under these conditions the magnetic splittings, like the effective g values observed by EPR, are very features, and we suspected that this redox-buffering ability sensitive to the rhombicity parameter E/D. Consequently, the high- was related to the functioning of the respiratory electron 3+ spin Fe ions in our sample produce broad and barely discernible transport chain. In an attempt to block this chain and thus features in weak fields. However, the 8.0-T spectra are fairly prevent cytochrome oxidase from reducing O , we treated a insensitive to D and E/D, because the large Zeeman splitting puts 2 3+ sample with antimycin A, a potent inhibitor of cytochrome essentially all Fe ions into the MS = 5/2 state, facilitating detection and quantification. bc1 [105], and then exposed it to O2. The spectrum of this 123 J Biol Inorg Chem (2007) 12:1029–1053 1041

a be improved by studying matched samples with both EPR b and Mo¨ssbauer spectroscopy, and we plan to do this in the 0.0 future.

0.2 A Discussion 0.4 The objectives of this study were to estimate (1) the concentration of Fe in mitochondria; (2) the distribution of that a Fe into various structural groups (heme, Fe/S clusters, etc.) b and (3) the degree to which the redox state of these groups Absorption (%) 0.0 could be altered by treating intact mitochondria with redox agents. Our approach was to obtain EPR and Mo¨ssbauer 0.2 B 8 T spectra of whole intact mitochondria. We consider the study to be exploratory and more qualitative than is nor- 0.4 mally the case for studies from our laboratories, for two essential reasons. First, it is impossible to determine metal concentrations of an organelle with anywhere near the -10 -5 0 5 10 precision typical of a purified metalloprotein. Second, it is Velocity (mm/s) impossible to deconvolute spectra into individual and Fig. 8 Mo¨ssbauer spectra at 4.2 K of dithionite-treated mitochondria assignable protein components. We explored whether at pH 8.5. Spectra were recorded in 50 mT (A) and 8.0 T (B) parallel obtaining even approximate and qualitative information applied fields. The dashed line outlines the contribution (45%) of relevant to these objectives would provide new insights species contained in the DEQ = 1.15 mm/s doublet. The solid lines + into how Fe is metabolized within these organelles. drawn above the data are spectral simulations of S = 1/2 [Fe4S4] cluster spectra using parameters of reduced aconitase (a 20%) and parameters similar to those of Escherichia coli sulfite reductase (b 20%). The solid lines drawn through the data are the sum of the three Metal and protein concentrations species. The rightmost feature in A is the high-energy line of a high- spin Fe2+ component. Its contribution, at 20%, is not taken into account in the simulation In order to determine the absolute concentration of proteins and Fe in whole intact mitochondria, we determined their concentration in packed mitochondria samples as well as sample (Fig. 7. spectrum D) exhibited the DEQ = 1.15 the fraction of the volume due to the mitochondria them- mm/s doublet with an intensity representing 52–57% of selves. Our calculations assumed that samples were devoid total 57Fe in the sample. The major difference between of impurities and that none of the radioactive sucrose used Fig. 7, spectra A and D is a decline from approximately in the experiment moved into the mitochondria. Sucrose is 20% in Fig. 7, spectrum A to 12% in Fig. 7, spectrum D of commonly used to match the osmotic pressure of mito- the high-spin Fe2+ species. An 8.0-T spectrum (not shown) chondrial buffers to that within the organelles, a property revealed that 5% of the total iron of the sample giving that implies the inability of sucrose to penetrate mito- Fig. 7, spectrum D is high-spin Fe3+. chondrial membranes. Electron micrographs and fluores- Because the central region of the spectrum of the cence images of our preparations did not reveal significant O2/antimycin A treated sample is comparatively clean, we contamination. The values obtained (approximately 800 lM studied this sample in an expanded velocity scale. This Fe by chemical analysis and approximately 500 lM 57Fe 2+ allowed us to search for the presence of [Fe2S2] and by Mo¨ssbauer spectroscopy, and approximately 70 mg/mL + [Fe2S2] clusters. Because of spectral crowding and the low protein) have relative uncertainties of about ±25%, as signal amplitudes, we do not have unequivocal evidence assessed from repeated measurements. for the presence of this cluster type, but we can give some As far as we are aware, all previously reported metal ion upper limits. We are quite confident that less than 5% of contents of mitochondria have been in terms of ratios of Fe 2+ the total iron belongs to (diamagnetic) [Fe2S2] clusters. concentrations to protein concentrations, typically given in Up to 12% of the iron may belong to conventional and units of nanomoles of Fe per milligram of mitochondrial + Rieske-type [Fe2S2] clusters, indicating that no more than protein (which in our case is approximately 10 nmol Fe/mg approximately 17% of the total iron in these samples arises protein). Given the complexity of the organelle, it may not from Fe2S2 clusters. Finally, only 8% of the total absorp- be possible to fully rationalize the ratio we obtained with + tion may belong to [Fe4S4] clusters. These estimates could other reported ratios. Cobine et al. [57] measured 2.3 nmol 123 1042 J Biol Inorg Chem (2007) 12:1029–1053

Fe/mg for mitochondria biosynthesized under respiratory located in the matrix (however, ferrochelatase receives conditions when the growth medium was not supplemented Fe2+ ions from the matrix). Henze and Martin [117] and with Fe, and 13 nmol Fe/mg when the medium was sup- Mu¨hlenhoff and Lill [118] suggest that the matrix is the plemented with Fe (our medium was not supplemented). It most anaerobic compartment in O2-respiring cells. Given does not appear that metal chelators were added during the predominant role of O2 in reactions occurring within isolation. The nearly sixfold observed difference suggests mitochondria, this might seem counterintuitive. However, that the Fe-to-protein concentration ratio depends sensi- the O2-consuming reactions occur at the IM which tively on the Fe concentration of the growth medium, and encapsulates the matrix, and these reactions may occur fast perhaps on other factors. Because these are ratios, differ- enough to effectively remove any O2 that diffuses into the ences may be due to changes in either Fe concentration matrix. + and/or protein concentration. Other reported values range In the oxidized inactivated state, the [Fe3S4] cluster of between 2.5 and 5 nmol Fe/mg mitochondrial protein aconitase affords an EPR signal with gave = 2.01 [5, 119], [106, 107]. Tangaras et al. [108] reported 4.3 nmol similar to that observed here. The Fe3S4 cluster of succ- Fe/mg, divided roughly into 20% heme, 50% Fe/S clusters cinate dehydrogenase exhibits a similar signal in the oxi- and 30% ‘‘non-heme non-FeS’’. Wallace et al. [6] reported dized state [2]. This signal was absent in all but one of the 1.2 nmol Fe/mg, while Kispal et al. [109] reported as-isolated samples, which may have been slightly oxidized 2 nmol/mg‘‘free’’ Fe (i.e., non-heme non-Fe/S). relative to other as-isolated samples. The more intense Fe3+ With use of in vivo fluorescence, the absolute concen- heme signals exhibited by this particular batch relative to tration of chelatable Fe within mitochondria from rat the other as-isolated samples are congruent with this pos- hepatocyte and endothelial cells has been estimated to be sibility. The absence of the gave = 2.01 signal in spectra of between 4.8 and approximately 12 lM[110, 111]. This our anaerobically prepared samples (and in spectra of should represent Fe on the inside of mitochondria but not dithionite-reduced mitochondria) as well as the presence of tightly associated with proteins. Mitochondria from human this signal under oxidizing conditions indicate that this + fibroblasts and lymphoblasts contain 1–2 lM of such signal probably arises from an oxidized [Fe3S4] cluster, chelatable Fe [112]. If similar concentrations of such Fe either from inactivated aconitase, homoaconitase or the + were present in our samples (approximately 8 lM), this [Fe3S4] cluster of succinate dehydrogenase. would represent only approximately 1% of the total Fe in these organelles; thus, we suspect that this is an underes- timate since we observe approximately 20% due to Adventitious Fe and Mn mononuclear high-spin Fe. We also caution that this refers to chelatable Fe within the organelle, and is not related to The hyperfine-split signal observed in the g = 2 region of the chelatable Fe which is responsible for the difference in various as-isolated samples is typical of adventitious or Fe concentrations observed with/without added chelators. weakly bound Mn2+ ions and we assign it as such. No additional Mn-based signals were observed in any of our samples, which suggests that the two known Mn-contain- Anaerobic isolation ing proteins in yeast mitochondria, Sod2p and Mtm1p, are present at concentrations below our EPR detection We isolated yeast mitochondria under anaerobic conditions limit. Sod2p is a matrix-localized manganese superoxide to afford better control of the redox status of these organ- dismutase while Mtm1p is an IM manganese chaperone elles. Anaerobic isolation was also a precautionary mea- [58]. Based on S = 5/2 spin Hamiltonian simulations using sure, because a number of mitochondrial proteins are the known parameters for Sod2p, namely, D = 0.348/cm inactivated by exposure to excess O2 or by the effects of and E/D = 0.026 [119, 121], our calculations indicate that oxidative stress. For example, iron is imported into the we could detect a minimum concentration of 10 lM Mn- matrix and delivered to the scaffold proteins in the reduced Sod2p. Thus, we suspect that Mn-Sod2p is present at a Fe2+ state [113]. Similarly, copper ions appear to be concentration less than this. imported in the reduced cuprous state. In vitro Fe/S bio- The g = 4.3 signal probably arises from Fe3+ ions that synthesis requires anaerobic conditions [114], biotin syn- are adventitiously bound to mitochondrial proteins or thase is inactivated by O2 [18] and maximal ferrochelatase membrane phospholipids. Our studies show that this type activity is observed under anaerobic conditions [115]. of iron can be removed by EDTA and EGTA, suggesting

Under oxidizing conditions, the labile iron in the Fe4S4 that it resides in a region that can be accessed by these cluster of aconitase dissociates into an Fe3S4 cluster, chelators, such as the outer face of the OM or perhaps the thereby inactivating the enzyme [116]. With the exception IMS. In more recent preparations, the amount of this 3+ of the IM ferrochelatase, these O2-sensitive enzymes are adventitious Fe appears to be minimal, even in samples 123 J Biol Inorg Chem (2007) 12:1029–1053 1043 prepared in the absence of chelators. The presence of cytochrome c, suggesting that it is in redox-equilibrium aqueous Fe3+ ions in the OM or the IMS suggests that this with the IMS. Assuming this, the presence of this signal region has an electrochemical potential sufﬁciently high to suggests that the potential of the IMS is less than +280 mV support this state. Previous reports have used the g = 4.3 in our samples. signal as a quantitative diagnostic for ‘‘free’’ Fe generated by cellular damage [121], but our study suggests that caution should be applied in these interpretations in that Species containing hemes this signal may also arise from Fe3+ peripherally associated with these organelles. Two of the observed signals which typify S = 5/2 Fe3+ hemes, including the g = (6.4, 5.4) signal with rhombic symmetry and the g = 6.0 signal with axial symmetry Species containing Fe/S clusters (E/D * 0) most likely arise from cytochrome c oxidase. Signals with identical g values have been reported to arise

The gave = 1.94 signal was a reproducible spectral feature from the heme a3:Cub active site in an intermediate redox of all as-isolated and dithionite-reduced samples. We ten- state in which Cub is reduced to the 1+ state, while heme a3 + 3+ tatively assign this signal to the [Fe2S2] cluster of succi- is high-spin Fe [70, 127, 128]. These signals are observed nate dehydrogenase. This cluster, when reduced, exhibits for samples that were oxidized by O2 or ferricyanide (and an EPR signal with g values nearly identical to those in spectra from the single ‘‘as-isolated’’ batch that exhib- observed here [123]. A similar signal in other reported ited the gave = 2.01 signal and was slightly more oxidized mitochondrial preparations has been observed and assigned than the others). The occurrence of this signal probably similarly [77]. However, the assignment should be cau- requires mildly oxidizing conditions, in that exposure to O2 tiously accepted because gave = 1.94 signals are charac- in our protocol was followed by a relatively slow anaerobic + + teristic of both [Fe4S4] and [Fe2S2] containing proteins packing procedure during which time some re-reduction and we cannot exclude the possibility that the observed could have occurred. The absence of these high-spin Fe3+ + signal arises from one or more [Fe4S4] clusters rather than, signals in the dithionite-treated samples is consistent with + 3+ or in addition to, the [Fe2S2] cluster in Sdh2p. the ability of dithionite to reduce Fe heme a3.This Further support for assigning the gave = 1.94 signal to the behavior is also consistent with a reduction potential for the + 3+ 2+ [Fe2S2] cluster of succinate dehydrogenase comes from the Fe /Fe heme a3 site of approximately +350 mV [129]. saturation properties of the gave =1.94signal(P1/2 =57mW In EPR studies of isolated cytochrome c oxidase, the at 10 K), which are similar to those reported for the succinate combined quantified intensity of these signals corre- + dehydrogenase [Fe2S2] cluster under conditions where the sponded to 23–50% of the cytochrome c oxidase concen- Fe4S4 cluster in the same enzyme is reduced to the para- tration [70, 127, 128]. Since the maximum combined spin magnetic 1+ state [3]. When the Fe4S4 cluster is oxidized to concentration observed here for these signals was approx- the diamagnetic 2+ state, the saturation behavior of the imately 3 lM, these percentages suggest a minimum + [Fe2S2] cluster differs substantially (P1/2 = 0.63 mW). The cytochrome c oxidase concentration in mitochondria of 6– observed saturation behavior suggests that the Fe4S4 cluster 13 lM. The combined heme a plus heme a3 concentration is reduced to the 1+ core oxidation state in our samples, and in mitochondria has been estimated at 0.15–0.3 lmol/g spectral features due to this species should be a component of mitochondrial protein [130], which suggests a cytochrome the Mo¨ssbauer spectra shown in Fig. 7. Our inability to c oxidase concentration (assuming 70 mg/mL protein + observe the broad EPR features reported for the [Fe4S4] concentration) of 5–10 lM, close to what we observe by cluster of Sdh2 is not surprising as these low-intensity fea- spin quantification. The intense signal that developed upon 0 2+/+ tures are easily missed [3]. Since E for the [Fe4S4] treating mitochondria with nitric oxide undoubtedly arose couple is 270 mV [124, 125], this implies that the potential from pentacoordinate heme–nitrosyl groups [104] and it of the solution for which this cluster is in redox-equilibrium indicates a minimum heme concentration in our samples of is at or below this value. 20 lM. A significant contribution to this signal is likely 2+ The gave = 1.90 signal is similar to that exhibited by the from NO binding to the Fe heme a3 of cytochrome c + isolated Rieske [Fe2S2] protein [4], and we assign it as oxidase. Considered collectively, we suspect that the con- such. It should contribute to the magnetic components of centration of cytochrome c oxidase in mitochondria from Fig. 7. E0 for the 2+/1+ redox couple of this cluster is respiring yeast is between 6 and 20 lM. 3+ +280 mV [126]. This protein is part of the cytochrome bc1 The third high-spin Fe heme signal (g = 6.8, 5.0; complex, which is located in the IM, but the Rieske protein E/D = 0.042) probably originates from heme b in cyto- itself is tethered to the rest of the complex and extends into chrome c peroxidase (Ccp1p), as similar g values have the IMS. This cluster transfers electrons to the IMS protein been reported [131, 132]. The particular degree of rhombic 123 1044 J Biol Inorg Chem (2007) 12:1029–1053 distortion in this heme depends on pH and subtle structural An unassigned mitochondrial EPR signal changes. The redox potential for the Fe3+/Fe2+ couple of this IMS protein is 182 mV at pH 7 [133]. If this signal Our samples exhibited an EPR signal with a distinct res- arises from Ccp1p, the potential of the IMS region in our onance at g = 2.08 and having gave = 2.02. Positive fea- samples would appear to be greater than this value. tures near g = 2.08 are usually associated with Fe4S4 Alternatively, this Fe3+ heme signal may originate from clusters, however our signal does not show the corre- matrix-localized catalase A (Cta1p) or flavohemoprotein sponding higher-field features typical of such clusters; (Yhb1p) as the Fe3+ states of these proteins exhibit similar rather, the partners for this species appear to be in the 2.00 g values [134, 135]. A similar signal from mitochondria region. Broader signals at or near g = 2.08 have been from Spodeptera littoralis was assigned to catalase [77]. assigned to a spin-coupled cluster involving the reduced S2 However, we expect that the potential of the matrix would cluster of complex II (succinate CoQ oxidoreductase) [3, be sufficiently low to reduce these centers fully. 142] and we have considered assigning it as such. We have EPR signals from low-spin Fe3+ hemes are typically also considered assigning it to ETF dehydrogenase [25– found at gz = 3.7–2.4 and gy = 2.5–2.1 [136], but no rec- 27]. Given the uncertainties, we leave this signal unas- ognizable signals were found in this region. Signals from signed pending further study. low-spin Fe3+ hemes were probably not observed either because such groups were in the Fe2+ state or because they are highly anisotropic with very broad signals. There are a Absence of Cu2+-based signals number of such groups in mitochondria (e.g., cytochromes 2+ b, b2, c, c1, heme a), many of which should be in redox- We did not observe signals characteristic of Cu ions, equilibrium with the IMS. Previous studies reported con- even though, by chemical analysis, our samples contained centrations of 0.2–0.4 lmol/g protein for cytochrome c and copper at detectably high concentrations. The lack of such

0.07–0.25 lmol/g protein for cytochrome c1 [130], both of signals suggests that the vast majority of Cu in our samples which correspond to easily detectable concentrations in our is in the diamagnetic Cu+ state. The most well-known Cu samples with 70 mg/mL protein. With an estimate of centers in mitochondria are the CuA and CuB sites in 0.8 lM for the concentration of flavocytochrome b2 cytochrome c oxidase. Oxidized CuA exhibits an EPR (Table 4), this protein would also be detectable. Since signal with g = 2.17 [143], but no such signal was obvi- 0 0 E = +290 mV for cytochrome c [137], +230 mV for ously present. E for CuA is +240 mV [144]. This center cytochrome c1 [138], 3 mV for flavocytochrome b2 [139] should be in redox-equilibrium with the IMS, as it func- and +255 mV for heme a [140], it seems likely that the tions by accepting electrons from cytochrome c. Since potential of the IMS in our samples was below approxi- there is a detectable concentration of cytochrome c oxidase mately 0 mV, rendering these centers ferrous and in our samples, this implies that the absence of a signal EPR-silent. arises because the potential of the IMS is less than approximately +230 mV. Mitochondria also contain a number of Cu chaperones, but such centers were probably Organic radical species in the diamagnetic Cu+ state [145, 146]. The majority of Cu in yeast mitochondria appears unassociated with proteins + The isotropic giso = 2.00 signal has g values and saturation and located in the matrix in a Cu form [57]. Our results properties typical of organic radicals, and we assign this are consistent with this, both in terms of the concentration signal to the population of such radicals in our samples. of Cu observed and the absence of Cu2+-based EPR signals. Possible sources include the semiquinone states of flavins However, we remain puzzled why no Cu2+ signals were and ubiquinone, and conceivably reactive oxygen species. observed under oxidizing conditions. Given the preponderance of ubiquinone (0.6–4.0 lmol/g mitochondrial protein) [141] and flavin-containing proteins (see ‘‘Introduction’’), we were surprised that the spin Electrochemical potentials of mitochondrial concentrations of the giso = 2.00 signal were so low. This compartments circumstance may have arisen because our mitochondria were isolated anaerobically such that any radical species It would be difficult to interpret our EPR results by generated during cell growth could have decayed during assuming that all redox centers in our mitochondrial sam- the lengthy isolation period. The increased intensity of the ples sensed the same electrochemical potential. Given the + giso = 2.00 signal for samples prepared under oxidizing likelihood that the [Fe2S2] cluster of succinate dehydro- conditions supports this possibility and highlights the genase was observed, with saturation properties indicating importance of preparing these organelles anaerobically. that the Fe4S4 cluster of this enzyme was also reduced, the 123 ilIogCe 20)12:1029–1053 (2007) Chem Inorg Biol J Table 4 Iron-containing proteins in mitochondria from S. cerevisiae Protein Gene Copies Concentration Location Prosthetic E0 (mV, NHE) Electronic and magnetic properties per cell (lM) group

Cytochrome c isoform I cyc1 7,730 1.2 IMS [46] Heme c +290 [137] S = 1/2 Fe3+ (g = 3.06, 2.26, 1.25) [32] and S =0Fe2+ Cytochrome c isoform II cyc7 1,310 0.2 IMS [46] Heme c +286 [156] S = 1/2 Fe3+ (g = 3.2, 2.05, 1.39) [32] and S =0 Fe2+ Cytochrome c peroxidase ccp1 6,730 1.1 IMS [157] Heme b 182 [133] S = 5/2 Fe3+ (g = 6.60, 5.23, 5-CN; and g = 6.13, 5.81, 6-CN) [158] and S =2Fe2+ 3+ Flavocytochrome b2 cyb2 4,590 0.8 IMS [157] Heme b2 3[139] S = 1/2 Fe (g = 2.99, 2.22, 1.47) [159] and S =0Fe2+ 2+ + Cytochrome bc1 rip1 – – IM (facing IMS) Fe2S2 (Rieske) +285 [126, 160] S = 0 [Fe2S2] and S = 1/2 [Fe2S2] (g = 2.02, [28, 155] 1.90, 1.80) [4] 3+ Cytochrome bc1 cyt1 39,900 6.6 IM (redox with Heme c1 +230 (est) [138] S = 1/2 Fe (g = 3.33 or 3.35) [161, 162] and rip1) [28, 155] low-spin Fe2+ Cytochrome c oxidase cox1 – – IM [163] Heme a +320 [31] S = 1/2 Fe3+ (g = 3.03, 2.21, 1.45) [127, 164] and low-spin Fe2+ 3+ Cytochrome c oxidase cox1 – – IM [163] Heme a3:Cub +350 [129] Fully oxidized: EPR-silent Fe spin-coupled to Cu2+ with J * 1/cm. Intermediate: S = 5/2 Fe3+ (g = 6.4, 5.3) [127] mixed with (g = 6.0) when Cu+. Fully reduced: high-spin Fe2+ :Cu+ [164, 165] Succinate dehydrogenase sdh3:sdh4 238:7,920 0.04:1.3 IM [1] Heme b +60 [166] (but this is for S = 1/2 Fe3+ (g = 3.63) [167] and S =0Fe2+ non-Sc enzyme which has novel Cys) 3+ 2+ Cytochrome bc1 cob1 – – IM [28, 155] Heme bH 45 (35 to +25) [155] S = 1/2 Fe (g = 3.45) [162] and S =0Fe 3+ 2+ Cytochrome bc1 cob1 – – IM [28, 155] Heme bL 150 (95) [155] S = 1/2 Fe (g = 3.78) [162] and S =0Fe 2+ Ferrochelatase hem15 22,700 3.8 IM (facing M) Mononuclear Fe – S =2Fe (d = 1.36 mm/s; DEQ = 3.04 mm/s) [42] [115] 2+ + Succinate dehydrogenase Sdh2 9,540 1.6 IM (facing M) Fe2S2 0[3] S = 0 [Fe2S2] and S = 1/2 [Fe2S2] (g = 2.026, [1] 1.935, 1.912) [3, 166] + 0 Succinate dehydrogenase Sdh2 9,540 1.6 IM (facing M) Fe3S4 +60 [3] S = 1/2 [Fe3S4] (g = 2.01) and S = 2 [Fe3S4] [1] [166] 2+ + Succinate dehydrogenase Sdh2 9,540 1.6 IM (facing M) Fe4S4 260 [3] S = 0 [Fe4S4] and S = 1/2 [Fe4S4] (g = 2.064, [1] 1.992, 1.847 and magnetic interactions affording features at 2.27 and 1.63) [3] Heme monooxygenase cox15 – – IM [35] Heme a [34, 168] +242 [168] S = 1/2 Fe3+ (g = 3.5) and S =0Fe2+ ,[168] Heme monooxygenase cox15 – – IM [35] Heme b [34, 168] +85 [168] S = 1/2 Fe3+ (g = 3.7) and S =0Fe2+ ,[168] Carboxylate monoxygenase Coq7 – – IM [169] Fe–O–Fe [48, 170] +48 and 135 [171] (Putative) S = 0 [Fe2+ Fe2+ ], S = 1/2 [Fe3+ Fe2+ ] 123 (g = 1.95, 1.86, 1.77) and S = 4 [Fe2+ Fe2+ ] [172]

2+ + 1045 ETF dehydrogenase YOR356W 3,320 0.6 IM Fe4S4 [26] +47 [27] S = 0 [Fe4S4] and S = 1/2 [Fe4S4] (g = 2.086, (putative) 1.939, 1.886) [25] 1046 123 Table 4 continued Protein Gene Copies Concentration Location Prosthetic E0 (mV, NHE) Electronic and magnetic properties per cell (lM) group

2+ + Aconitase Aco1 96,700 16 M [173]Fe4S4 and Fe3S4 450, 268, +100 [174] S = 0 [Fe4S4] and S = 1/2 [Fe4S4] (g = 2.06, + 1.93, 1.86) [5] S = 1/2 [Fe3S4] (g = 2.024, 0 2.016, 2.004) and S = 2 [Fe3S4] S = 1/2 3+ 2+ [Fe4S4] and S = 0 [Fe4S4]

Homoaconitase Lys4 7,350 1.2 M [6]Fe4S4 and Fe3S4 Similar to aconitase [6] Similar to aconitase [6] (putative) 2+ + Ferredoxin Yah1 14,800 2.4 M [7]Fe2S2 353 [8] S = 0 [Fe2S2] and S = 1/2 [Fe2S2] (g = 2.024, 1.937, 1.937) [8] 2+ Fe/S scaffold protein Isu1 10,800 1.8 M [22]Fe2S2 (Probably low) S = 0 [Fe2S2] [175] 2+ Fe/S scaffold protein Isu2 3,420 0.6 M [22]Fe2S2 (Probably low) S = 0 [Fe2S2] [175] 2+ Fe/S scaffold protein Isa1 125 0.02 M [23]Fe2S2 (Probably low) S = 0 [Fe2S2] [175] 2+ Fe/S scaffold protein Isa2 1,560 0.3 M [174] or IMS Fe2S2 (putative) (Probably low) S = 0 [Fe2S2] [175] [23] [176] 2+ Fe/S scaffold protein Nfu1 11,300 1.9 M [22]Fe2S2 (Probably low) S = 0 [Fe2S2] [175] 2+ Fe/S scaffold protein Isu1 10,800 1.8 M [22]Fe4S4 (Probably low) S = 0 [Fe4S4] [175] 2+ Fe/S scaffold protein Isu2 3,420 0.6 M [22]Fe4S4 (Probably low) S = 0 [Fe4S4] [175] 2+ Fe/S scaffold protein Isa1 125 0.02 M [23]Fe4S4 (Probably low) S = 0 [Fe4S4] [175] 2+ Fe/S scaffold protein Isa2 1,560 0.3 M [176] or IMS Fe4S4 (putative) (Probably low) S = 0 [Fe4S4] [175] [23] [176] 2+ Fe/S scaffold protein Nfu1 11,300 1.9 M [22]Fe4S4 (Probably low) S = 0 [Fe4S4] [175] 2+ + Biotin synthase Bio2 504 0.08 M [177]Fe4S4 440 [178] S = 0 [Fe4S4] and S = 1/2 [Fe4S4] (g = 2.042, 1.937, 1.937) [18]or(g = 2.035, 1.937, 1.937) [14]or(g = 2.044, 1.944, 1.914 and S = 3/2) [179] 2+ + Lipoic acid synthase Lip5 1,630 0.3 M [180]Fe4S4 505 [181] S = 0 [Fe4S4] and S = 1/2 [Fe4S4] (g = 2.039, 1.937, 1.937) [18]

2+ + 12:1029–1053 (2007) Chem Inorg Biol J Biotin synthase Bio2 504 0.08 M [177]Fe2S2 140 [178] S = 0 [Fe2S2] and S = 1/2 [Fe2S2] (g = 2.01, 1.96, 1.88 and g = 2.00, 1.94, 1.85) [14, 182] 2+ + Lipoic acid synthase Lip5 1,630 0.3 M [180]Fe2S2 430 [181] S = 0 [Fe2S2] and S = 1/2 [Fe2S2] 2+ + Dihydroxyacid dehydratase Ilv3 171,000 28 M (putative) [19]Fe4S4 (putative) (Dithionite-reducible) S = 0 [Fe4S4] and S = 3/2 [Fe4S4] (g = 5.2, [20] 4.7) [19, 20] Frataxin homolog Yfh1 1,560 0.3 M [183] 2 mononuclear Fe’s (Probably high) S = 5/2 Fe3+ and Fe2+ [184, 185] [184] Catalase A Cta1 623 0.1 M [37] Heme b 226 (est [134]) S = 5/2 Fe3+ (g = 6.48, 5.10) [186] Flavohemoglobin Yhb1 13,000 2.2 M [135] (and Heme b 230 to 320 (est [187]) S = 5/2 Fe3+ (g = 5.75, 6.47, 5.22) [188] cytosol) NHE normal hydrogen electrode, IMS intermembrane space, IM inner membrane, M matrix J Biol Inorg Chem (2007) 12:1029–1053 1047 potential of the solution with which this cluster is in redox- clusters. Spectra recorded in strong applied fields distin- equilibrium should be less than approximately 270 mV. guish also between monomeric high-spin Fe3+ and nano- These clusters of succinate dehydrogenase should be in particles containing high-spin Fe3+. redox-equilibrium with the mitochondrial matrix, which implies a matrix potential in our samples of less than 270 mV. We observe an oxidized Fe3+ heme which is Approximate iron distribution in mitochondria most likely from the IMS cytochrome c peroxidase, and the from respiring yeast lack of a signal from flavocytochrome b2 suggests that it is in the reduced state. This implies an IMS solution potential Our Mo¨ssbauer spectroscopy and EPR results are insuffi- in the range from 200 to 0 mV in our samples. If we cient to establish precisely how Fe ions in mitochondria are assume a potential difference between the IMS and the distributed, but they are sufficient to allow us to draw some matrix of 180 mV [147], an IMS potential range of 100 approximate and preliminary conclusions, which are sum- to 200 mV would predict a matrix potential range of marized by the pie chart shown in Fig. 9. The majority of Fe 300 to 400 mV, which are both compatible with our in mitochondria from respiring yeast is present as Fe4S4 observations. clusters; in Fig. 9 we estimate this to be approximately 60%, but values as low as 50% and as high as 85% are

possible. In the as-isolated state, most of these Fe4S4 clus- Mo¨ssbauer spectroscopy ters are in the 2+ state, but a substantial fraction can be reduced to the 1+ state by incubation of mitochondria with To date, only one Mo¨ssbauer spectrum of wild-type mito- dithionite at pH 8.5. Thus, in Fig. 9 we distinguish dithio- 2+/+ chondria has been published and it was devoid of any nite-reducible [Fe4S4] clusters from irreducible 2+ signals [39]. In contrast, a sample of the mutant Dyfh1 [Fe4S4] clusters. The next most abundant class of Fe- displayed a quadrupole doublet with DEQ = 0.67 mm/s and containing species in mitochondria, representing approxi- d = 0.52 mm/s which was assigned to amorphous nano- mately 20% of the Fe in Fig. 9 (but with an acceptable range particles of iron(III) phosphate; other Fe-containing components were not observed. We have recorded Mo¨ssbauer spectra of more than 25 preparations of intact mitochondria, over the course of 2 years and involving various group members preparing these samples. Also, the Mo¨ssbauer spectroscopy and EPR studies developed indepen- dently, and we have therefore not studied aliquots of the matched samples with both techniques. Isolation proce- dures were adjusted based on feedback from our Mo¨ssbauer spectroscopy and EPR results, so it is not surprising that we observed some variation in the concentration of the various spectroscopic components. For studies of mitochondria, EPR and Mo¨ssbauer spectroscopy are complementary. EPR detects with high sen- sitivity species with half-integral spin (Kramers systems), while Mo¨ssbauer spectroscopy, in this first study, is mainly useful in the exploration of components with integer or Fig. 9 Comparison of observed and calculated percentile Fe distribution. Percentages used in the pie chart for the observed distribution zero electronic spin, i.e., components either not accessible 2+ 2+ were as follows: 37% ([Fe4S4] + low-spin Fe hemes), 25% (diamagnetic complexes) or only difficult to access by 2+/+ 3+ 2+ [Fe4S4] , 22% high-spin (Fe +Fe ) non-heme mononuclear, 9% ¨ 2+/+ 3+ 2+ +/0 EPR. We consider our present Mossbauer spectroscopy [Fe2S2] , 4% high-spin hemes (Fe +Fe ) and 3% [Fe3S4] results to be preliminary, but we believe that the proven clusters. These values are based on our results, collectively consid- power of the technique can be exploited in the future, once ered, but should be viewed as a hypothesis, with substantial latitude in our estimates for each category. Calculated percentages were 78% the system is dissected by metabolic and/or genetic 2+/+ 2+ 3+/2+ [Fe4S4] , 5% low-spin hemes (Fe only), 5% high-spin Fe 2+/+ 3+/2+ manipulations, e.g., overexpression or deletion of particu- non-heme mononuclear, 8% [Fe2S2] , 2% high-spin hemes (Fe ) +/0 lar mitochondrial proteins. Our present studies suggest that and 2% [Fe3S4] . These values are taken from Table S1, assuming 2+ one should conduct the Mo¨ssbauer spectroscopy studies at 8 lM nonproteinatious high-spin non-heme Fe ions. The concen- 4.2 K and in weak as well as in strong applied fields. A tration of Fe associated with each Fe-containing mitochondrial protein was calculated and percentages were obtained by dividing strong-field capability is essential as it allows one to each individual Fe concentration by the sum of all such values and 2+ 2+ identify S = 0 species such as [Fe4S4] and [Fe2S2] multiplying by 100. HS low spin, LS low spin 123 1048 J Biol Inorg Chem (2007) 12:1029–1053 of 15–35%), is high-spin non-heme Fe. In the as-isolated Comparison with known Fe-containing proteins state, most of these ions are high-spin Fe2+, with smaller in mitochondria proportions of high-spin Fe3+. We have not observed low- spin Fe3+ ions in any of our samples. Our EPR results We have organized known mitochondrial proteins start- suggest that approximately 9% (but with a range of 5–11%, ing with the results of three proteomic studies [148–150], as limited by our Mo¨ssbauer spectroscopy analysis) of the the information provided by the Saccharomyces Ge- + Fe is present as [Fe2S2] clusters, whereas no such clusters nomics Database and the reconstructed metabolic net- in the 2+ state were observed. The DEQ = 1.15 mm/s work of Forster et al. [151]. The integration of this 2+ doublet, attributed essentially to [Fe4S4] clusters, may information led to the identification of approximately 600 contain contributions from low-spin ferrous hemes. The candidate mitochondrial proteins, a number comparable remaining few percent of mitochondrial Fe is present as to the approximately 800 proteins estimated to constitute high-spin heme and perhaps Fe3S4 centers. the complete yeast mitochondrial proteome [150]. The spectral simulations of Fig. 8 suggest that the main Primary research literature describing properties of each + part of the magnetic features represent S = 1/2 [Fe4S4] of these proteins was accessed using the Web of Science clusters. While we are reasonably certain that the magnetic (http://www.isi10.isiknowledge.com) and information features reflect paramagnetic Fe/S clusters, we do not wish specifically regarding Fe content and suborganellar to suggest that the entire magnetic component belongs to localization was sought. + the S = 1/2 forms (the gave = 1.94 species) of [Fe4S4] The result of this analysis afforded the proteins and pro- + clusters; we suspect that S = 3/2 [Fe4S4] as well as tein complexes included in Table 4. It is difficult to establish + [Fe2S2] clusters, albeit to a lesser extent, also contribute. that this or any such list is complete, and we suspect that By EPR, we see signals assigned to these latter species there are Fe-containing mitochondrially localized proteins + + (S = 3/2 [Fe4S4] and S = 1/2 [Fe2S2] clusters). By that are not included. Some such proteins might be uniden- studying the Mo¨ssbauer and EPR spectra of aliquots of the tified currently, or the presence of Fe in them might be same dithionite-treated sample, one should be able to uncertain. Also not included in this list are proteins that are assess the cluster type and concentration of the reduced Fe/ known to interact with Fe (e.g., transporters) but for which S species better. no Fe-bound state has been characterized. Regarding high-spin ferrous species, many hexa-and The concentrations of many of these proteins within the pentacoordinated complexes with N/O ligation contribute mitochondria have been estimated. Ghaemmaghami et al. doublets with DEQ = 3.0–3.5 mm/s and d = 1.3–1.4 mm/s. [153] created a comprehensive fusion library of S cerevisiae With a few exceptions the high-field spectra of these cells in which each member had a different open reading complexes are broad and difficult to analyze. frame tagged with the same epitope. Natural expression 2+ Included in the [Fe4S4] cluster portion are clusters that levels of the corresponding fusion proteins were quantified convert into Fe3S4 clusters upon oxidation (e.g., aconitase); to afford copy numbers per cell (Table 4). The volume of an this fraction could represent as much as approximately S. cerevisiae cell is approximately 1 · 1013 L, and mito- 25 lM Fe (3% of the total). Treatment with dithionite at chondria occupy approximately 10% of this [154]. Thus, 2+ pH 8.5 causes approximately half of the [Fe4S4] portion one copy of a mitochondrial component per cell reflects a + to become reduced to the [Fe4S4] state. concentration of 170 pM in the organelle. Such information Mo¨ssbauer spectra of as-isolated mitochondria indicate can be useful in interpreting Mo¨ssbauer spectra. that approximately 15% of the Fe is paramagnetic and in It is interesting to compare this list with the results half-integer spin states. According to our EPR results, this observed in this study. We calculated the overall Fe con- would include approximately 3 lM due to the high-spin centration in mitochondria implied by the proteins and heme signal from cytochrome c peroxidase/catalase, concentrations given in this table, by summing the products approximately 3 lM due to the high-spin heme signals of the concentration of each known Fe-containing mito- from cytochrome c oxidase, approximately 20 lM due to chondrial protein (Ci)P and the number of Fe’s per protein 3+ n g = 4.3 high-spin Fe , approximately 40 lM due to the (mi); i.e., [Fe]overall ¼ i¼1 miCi (if the number of copies + [Fe2S2] cluster of the Rieske protein and approximately per cell was not reported, a concentration of 1 lM was + 20 lM due to the [Fe2S2] cluster from succinate dehy- assumed). The calculated overall concentration of Fe in + drogenase. The [Fe4S4] cluster of succinate dehydroge- mitochondria was 265 lM (Table S1), corresponding to nase might also contribute to the paramagnetic component. only one third of our experimentally determined value. If Summing these EPR contributions affords approximately correct, this suggests that two thirds of the detected Fe in 90 lM Fe, translating into approximately 11% of the total yeast mitochondria is not accounted for by Table 4, Fe, in qualitative agreement with what is observed by assuming the protein concentrations in that table. On the Mo¨ssbauer spectroscopy. other hand, systematically low estimates of protein 123 J Biol Inorg Chem (2007) 12:1029–1053 1049

concentration in the literature might also be responsible for respectively (see discussion above). EIM probably lies this discrepancy. Tempered by this caveat, some interesting somewhere between these two values and may be con- trends are apparent, namely: trolled by the E value for the CoQox/CoQred couple, namely, +60 mV in the IM [155]. What must separate the • A large proportion of Fe in mitochondria appears to be redox potential of one region from another and maintain associated with the Fe S cluster from a single protein, 4 4 regions in redox isolation are the redox-dependent con- namely, dihydroxyacid dehydratase. formational changes known for the IM-bound respiratory • The OM is devoid of Fe-containing proteins. complexes. • The matrix contains few heme-containing proteins Then, we grouped all species of Table 4 that would give (only catalase and flavohemoglobin). rise to equivalent Mo¨ssbauer spectral features, and calcu- • The matrix is dominated by Fe/S centers, especially lated the concentration of Fe associated with each group. [Fe S ]2+ clusters. 4 4 This procedure resulted in the nine groups listed in • Only one mitochondrial protein with an Fe–O–Fe Table S1. The calculated percentages of mitochondrial Fe center is known (Coq7p). in various forms are also shown in Fig. 9. Comparison with In order to compare our experimental EPR and Mo¨ssbauer what we have observed in this study indicates overall spectroscopy results with predictions made from these qualitative agreement. The greatest apparent discrepancy is calculations, two additional pieces of information for each the greater percentage of high-spin non-heme Fe observed site are required, namely, the region of the mitochondria in our samples relative to that predicted by the calculations. with which the site is in redox-equilibrium and the solution The qualitative similarities in the calculated versus potential of that region under the conditions when our observed distribution of Fe in mitochondria indicates a samples were frozen. For some redox centers (e.g., cyto- general agreement between our results and the calculated chrome c), there is no doubt as to the region with which they contents of these organelles. Calculations predict that there 2+ 2+ are in redox-equilibrium (e.g., the IMS), but such informa- should be fewer high-spin Fe ions and more ([Fe4S4] tion is not certain for all entries in Table 4. Nor is the clusters + low-spin Fe2+ hemes) than we observe. Some of solution potential for each region of the mitochondria the observed high-spin Fe2+ ions may be adventitiously (under the specific conditions for which our samples were bound, despite our attempt to remove such ions by chela- prepared) known. For proteins located in either aqueous tion. Alternatively, some Fe-containing mitochondrial region (IMS or matrix), the region with which they were proteins have not been included in the calculations, or a assumed to be in redox-equilibration was the region where portion of these ions might represent a transient and che- the proteins were located. For proteins located in the IM, latable ferrous ion pool, as has been reported previously there were a number of possibilities. Some sites extend into [111]. In this latter case, the concentration of ions esti- either the IMS or the matrix, and if such sites are known to mated for this pool (2–12 lM Fe) would be substantially accept/donate electrons with donors/acceptors in that less than we observe (approximately 180 lM). aqueous region, such centers were deemed to be in redox- equilibrium with that aqueous region rather than with the IM. Other sites contained within IM-bound respiratory Conclusions complexes might be along a known electron pathway which leads to either aqueous region, and such sites were deemed The major contributions of this study are: to be in redox-equilibrium with that aqueous region. In a few cases, redox-dependent protein conformation changes 1. Protein and metal concentrations. We determined the occur such that sites contained within those proteins might absolute concentrations of protein and Fe in ‘‘neat’’ be in redox-equilibrium with more than one region, (solvent-free) yeast mitochondria, synthesized by depending on the protein conformation at the time our respiring cells and isolated in the presence of metal samples were frozen, and so no definitive assignment could chelators to be approximately 70 mg/mL and 800 ± be made. Finally, the active site for cytochrome c oxidase 200 lM, respectively.

(heme a3:Cub) might be unique in being in redox-equilib- 2. EPR signals from proteins containing Fe/S cluster and rium with the O2/H2O couple (E * +800 mV). For our heme prosthetic groups. Signals were observed that samples prepared under anaerobic conditions, the appro- have been tentatively assigned to succinate dehydro- priate non-standard-state reduction potential to use would genase, the Rieske Fe/S protein, aconitase, cytochrome be much less than +800 mV. Our set of tentative assign- c oxidase and cytochrome c peroxidase. An intense ments is given in Table S1. signal with gave = 2.02 was observed. Although Next, we estimated the solution potential of the IMS and unassigned, this signal probably originates from an matrix to be EIMS & 0.1 and Ematrix & 0.3 V, Fe/S cluster. 123 1050 J Biol Inorg Chem (2007) 12:1029–1053

3. Species not observed. No signals from Cu2+ ions, Mn2+ 3. Maguire JJ, Johnson MK, Morningstar JE, Ackrell BAC, superoxide dismutase or low-spin Fe3+ hemes were Kearney EB (1985) J Biol Chem 260:10909–10912 4. Fee JA, Findling KL, Yoshida T, Hille R, Tarr GE, Hearshen observed. These species may be present at undetecta- DO, Dunham WR, Day EP, Kent TA, Mu¨nck E (1984) J Biol bly low concentrations or in EPR-silent states. The Chem 259:124–133 collective concentration of organic-based radical spe- 5. Emptage MH, Kent TA, Kennedy MC, Beinert H, Munck E cies was unexpectedly low. (1983) Proc Natl Acad Sci USA 80:4674–4678 6. Wallace MA, Liou LL, Martins J, Clement MHS, Bailey S, 4. Electrochemical compartmentalization. In the as-iso- Longo VD, Valentine JS, Gralla EB (2004) J Biol Chem lated state of our samples, the ranges of the potentials 279:32055–32062 for the IMS and the matrix are 0.2 < EIMS < 0.1 V 7. Barros MH, Nobrega FG (1999) Gene 233:197–203 8. Schifﬂer B, Bureik M, Reinle W, Muller EC, Hannemann F, and 0.4 < Ematrix < 0.3 V (vs NHE), respectively. Bernhardt R (2004) J Inorg Biochem 98:1229–1237 5. Dominance of Fe4S4 clusters. The majority of Fe (50– 9. Xia B, Cheng H, Bandarian V, Reed GH, Markley JL (1996) 85%) in mitochondria isolated from respiring cells is Biochemistry 35:9488–9495 present in this form. 10. Mitou G, Higgins C, Wittung-Stafshede P, Conover RC, Smith 6. High-spin ferrous ions. Approximately 20% of the Fe AD, Johnson MK, Gaillard J, Stubna A, Mu¨nck E, Meyer J 2+ (2003) Biochemistry 42:1354–1364 in mitochondria is present as high-spin non-heme Fe 11. Zhang S, Sanyal I, Bulboaca GH, Rich A, Flint DH (1994) Arch ions with ﬁve to six O/N ligands coordinating. A Biochem Biophys 309:29–35 portion of this is probably adventitiously bound to the 12. Berkovitch F, Nicolet Y, Wan JT, Jarrett JT, Drennan CL (2004) mitochondria, while the remainder is associated with Science 303:76–79 13. Cosper MM, Jameson GNL, Eidsness MK, Huynh BH, Johnson mitochondrial proteins and/or conceivably a ‘‘pool’’ of MK (2002) FEBS Lett 529:332–336 such ions, perhaps in the mitochondrial matrix. 14. Jameson GN, Cosper MM, Hernandez HL, Johnson MK, Huynh 7. Fe2S2 clusters. A modest proportion of the Fe BH (2004) Biochemistry 43:2022–2031 (approximately 5–10%) in mitochondria is present as 15. Hewitson KS, Ollagnier-de Choudens S, Sanakis Y, Shaw NM, Baldwin JE, Mu¨nck E, Roach PL, Fontecave M (2002) J Biol these types of clusters, including those from succinate Inorg Chem 7:83–93 dehydrogenase and the Rieske Fe/S protein. 16. Kriek M, Peters L, Takahashi Y, Roach PL (2002) Protein Expr 8. Hemes. A similarly modest proportion of Fe in Purif 28:241–245 mitochondria is present as heme prosthetic groups. 17. Miller JR, Busby RW, Jordan SW, Cheek J, Henshaw TF, Ashley GW, Broderick JB, Cronan JE, Marletta MA (2000) 9. Distribution of Fe in mitochondrial regions. The OM Biochemistry 39:15166–15178 is largely devoid of Fe-containing proteins, while the 18. Ollagnier-de Choudens S, Sanakis Y, Hewitson KS, Roach P, matrix is dominated by Fe4S4 clusters. Baldwin JE, Mu¨nck E, Fontecave M (2000) Biochemistry 39:4165–4173 19. Velasco JA, Cansado J, Pena MC, Kawakami T, Laborda J Note added in proof: It now appears less likely that Isa1p and Isa2p (1993) Gene 137:179–185 contain Fe/S clusters similar to those in other Fe/S scaffold proteins. 20. Flint DH, Emptage MH, Finnegan MG, Fu WG, Johnson MK (1993) J Biol Chem 268:14732–14742 Acknowledgements We thank the following people: Art Johnson 21. Mu¨hlenhoff U, Gerber J, Richhardt N, Lill R (2003) EMBO J and Holly Cargill (Department of Biochemistry and Biophysics, 22:4815–4825 Texas A&M University) for instructions on isolating mitochondria; 22. Schilke B, Voisine C, Beinert H, Craig E (1999) Proc Natl Acad Rola Barhoumi (Image Analysis Laboratory, Texas A&M University) Sci USA 96:10206–10211 and Anne Ellis (Microscopy and Imaging Center, Texas A&M Uni- 23. Jensen LT, Culotta VC (2000) Mol Cell Biol 20:3918–3927 versity) for collecting microscopic images; Jinny Johnson (Protein 24. Tong WH, Jameson GNL, Huynh BH, Rouault TA (2003) Proc Chemistry Laboratory, Texas A&M University) for performing amino Natl Acad Sci USA 100:9762–9767 acid analyses; David P. Giedroc (Department of Biochemistry and 25. Ruzicka FJ, Beinert H (1975) Biochem Biophys Res Comm Biophysics, Texas A&M University) for access to his atomic 66:622–631 absorption spectrophotometer; William James (Department of 26. Ruzicka FJ, Beinert H (1975) J Biol Chem 252:8440–8445 Chemistry, Texas A&M University) for training on and assistance 27. Paulsen KE, Orville AM, Frerman FE, Lipscomb JD, Stanko- with the inductively coupled plasma mass spectrometer; Shelly vich MT (1992) Biochemistry 31:11755–11761 Henderson Possi for help in isolating some batches and in measuring 28. Hunte C, Koepke J, Lange C, Rossmanith T, Michel H (2000) O2 consumption; Tanner Freeman for preparing one of the EPR Structure 8:669–684 samples; and Roland Lill for helpful discussion.This study was sup- 29. Bonagura CA, Bhaskar B, Shimizu H, Li HY, Sundaramoorthy ported by the Robert A. Welch Foundation (A1170) and The National M, McRee DE, Goodin DB, Poulos TL (2003) Biochemistry Institutes of Health [GM077387 (M.P.H.), EB001475 (E.M.) and The 42:5600–5608 Chemistry Biology Interface training program (B.N.H. and J.G)]. 30. Mowat CG, Miles CS, Munro AW, Cheesman MR, Quaroni LG, Reid GA, Chapman SK (2000) J Biol Inorg Chem 5:584– 592 References 31. Kojima N, Palmer G (1983) J Biol Chem 258:4908–4913 32. Brautigan DL, Feinberg BA, Hoffman BM, Margoliash E, 1. Lemire BD, Oyedotun KS (2002) Biochim Biophys Acta Bio- Peisach J, Bumberg WE (1977) J Biol Chem 252:574–582 energ 1553:102–116 33. Barros MH, Carlson CG, Glerum DM, Tzagoloff A (2001) 2. Beinert H (2002) Biochim Biophys Acta Bioenerg 1553:7–22 FEBS Lett 492:133–138

123 J Biol Inorg Chem (2007) 12:1029–1053 1051

34. Hederstedt L, Lewin A, Throne-Holst M (2005) J Bacteriol 69. Oxelmark E, Marchini A, Malanchi I, Magherini F, Jaquet L, 187:8361–8369 Hajibagheri MAN, Blight KJ, Jauniaux JC, Tommasino M 35. Glerum DM, Muroff I, Jin C, Tzagoloff A (1997) J Biol Chem (2000) Mol Cell Biol 20:7784–7797 272:19088–19094 70. Sands RH, Beinert H (1960) Biochem Biophys Res Commun 36. Bureik M, Schifﬂer B, Hiraoka Y, Vogel F, Bernhardt R (2002) 3:47–52 Biochemistry 41:2311–2321 71. Hartzell CR, Beinert H (1974) Biochim Biophys Acta 368:318– 37. Petrova VY, Drescher D, Kujumdzieva AV, Schmitt MJ (2004) 338 Biochem J 380:393–400 72. Bulteau AL, Ikeda-Saito M, Szweda LI (2003) Biochemistry 38. Ding H, Harrison K, Lu J (2005) J Biol Chem 280:30432– 42:14846–14855 30437 73. Lin CIP, Ohnishi T, Clejan L, Beattie DS (1983) Eur J Biochem 39. Lesuisse E, Santos R, Matzanke BF, Knight SAB, Camadro JM, 137:179–183 Dancis A (2003) Hum Mol Genet 12:879–889 74. Phillips JD, Graham LA, Trumpower BL (1993) J Biol Chem 40. Sellers VM, Wu CK, Dailey TA, Dailey HA (2001) Biochem- 268:11727–11736 istry 40:9821–9827 75. Merbitz-Zahradnik T, Zwicker K, Nett JH, Link TA, Trum- 41. Ferreira GC, Franco R, Mangravita A, George GN (2002) Bio- power BL (2003) Biochemistry 42:13637–13645 chemistry 41:4809–4818 76. Shergill JK, Cammack R (1994) Biochim Biophys Acta 42. Wu CK, Dailey HA, Rose JP, Burden A, Sellers VM, Wang BC 1185:43–49 (2001) Nat Struct Biol 8:156–160 77. Shergill JK, Cammack R, Chen JH, Fisher MJ, Madden S, Rees 43. Foury F, Roganti T (2002) J Biol Chem 277:24475–24483 HH (1995) Biochem J 307:719–728 44. Li LT, Kaplan J (1997) J Biol Chem 272:28485–28493 78. Albract SPJ, Subramanian J (1977) Biochim Biophys Acta 45. Moraes CT, Diaz F, Barrientos A (2004) Biochim Biophys Acta 462:36–48 Bioenerg 1659:153–159 79. Gavin CE, Gunter KK, Gunter TE (1999) Neurotoxicology 46. Dumont ME, Cardillo TS, Hayes MK, Sherman F (1991) Mol 20:445–453 Cell Biol 11:5487–5496 80. Guldutuna S, Zimmer G, Leuschner M, Bhatti S, Elze A, Bei- 47. Chloupkova M, LeBard LS, Koeller DM (2003) J Mol Biol singer B, Hofmann M, Leuschner U (1999) Biochim Biophys 331:155–165 Acta 1453:396–406 48. Lill R, Kispal G (2001) Res Microbiol 152:331–340 81. Tuckey RC, McKinley AJ, Headlam MJ (2001) Eur J Biochem 49. Lange H, Lisowsky T, Gerber J, Mu¨hlenhoff U, Kispal G, Lill R 268:2338–2343 (2001) EMBO Rep 2:715–720 82. Albracht SPJ, Leeuwerik FJ, VanSwol B (1979) FEBS Lett 50. Stenmark P, Gru¨nler J, Mattsson J, Sindelar PJ, Nordlund P, 104:197–200 Berthold DA (2001) J Biol Chem 276:33297–33300 83. Albracht SPJ (1980) Biochim Biophys Acta 612:11–28 51. Berthold DA, Voevodskaya N, Stenmark P, Gra¨slund A, 84. Albracht SPJ, VanVerseveld HW, Hagen WR, Kalkman ML Nordlund P (2002) J Biol Chem 277:43608–43614 (1980) Biochim Biophys Acta 593:173–186 52. Neese F, Zumft WG, Antholine WE, Kroneck PMH (1996) 85. Diekert K, de Kroon AIPM, Kispal G, Lill R (2000) In: Pon LA, J Am Chem Soc 118:8692–8699 Schon EA (eds) Methods in yeast genetics, vol 65. Academic, 53. Barros MH, Johnson A, Tzagoloff A (2004) J Biol Chem San Diego 279:49883–49888 86. Cleland WW (1964) Biochemistry 3:480 54. Palumaa P, Kangur L, Voronova A, Sillard R (2004) Biochem J 87. Barondeau DP, Roberts LM, Lindahl PA (1994) J Am Chem Soc 382:307–314 116:3442–3448 55. Hiser L, Di Valentin M, Hamer AG, Hosler JP (2000) J Biol 88. Wright R (2000) Microsc Res Tech 51:496–510 Chem 275:619–623 89. Pelley JW, Garner CW, Little GH (1978) Anal Biochem 56. Sturtz LA, Diekert K, Jensen LT, Lill R, Culotta VC (2001) 86:341–343 J Biol Chem 276:38084–38089 90. Toyoshima S, Wantanabe F, Saido H, Miyatake K, Nakano Y 57. Cobine PA, Ojeda LD, Rigby KM, Winge DR (2004) J Biol (1995) J Nutr 125:2846–2850 Chem 279:14447–14455 91. Sapan CV, Lundlad RL, Price NC (1999) Biotechnol Appl 58. Luk E, Jensen LT, Culotta VC (2003) J Biol Inorg Chem 8:803– Biochem 29:99–108 809 92. Dineley KE, Richards LL, Votyakova TV, Reynolds IJ (2005) 59. Hirabaya T, Harada T (1971) Biochem Biophys Res Commun Mitochondria 5:55–65 45:1369 93. Lund P, Wiggins D (1990) Biochim Biophys Acta 1018:98–102 60. Sharp RE, White P, Chapman SK, Reid GA (1994) Biochem- 94. Hackenbrock CR (1968) J Cell Biol 37:345–369 istry 33:5115–5120 95. Zischka H, Weber G, Weber PJA, Posch A, Braun RJ, Bu¨hringer 61. Arscott LD, Gromer S, Schirmer RH, Becker K, Williams CH D, Schneider U, Nissum M, Meitinger T, Uefﬁng M, Eckerskorn (1997) Proc Natl Acad Sci USA 94:3621–3626 C (2003) Proteomics 3:906–916 62. Staples CR, Ameyibor E, Fu WG, Gardet-Salvi L, Stritt-Etter 96. Shaw JM, Nunnari J (2002) Trends Cell Biol 12:178–184 EL, Knaff DB, Johnson MK (1996) Biochemistry 35:11425– 97. Egner A, Jakobs S, Hell SW (2002) Proc Natl Acad Sci USA 11434 99:3370–3375 63. Garrib A, McMurray WC (1986) J Biol Chem 261:8042–8048 98. Polcic P, Sabova L, Kolarov J (1997) FEBS Lett 412:207–210 64. Huh WK, Kim ST, Yang KS, Seok YJ, Hah YC, Kang SO 99. Soubannier V, Vaillier J, Paumard P. Coulary B, Schaeffer J, (1994) Eur J Biochem 225:1073–1079 Velours J (2002) J Biol Chem 277:10739–10745 65. Joo HS, Kim SS (1998) J Biochem Mol Biol 31:37–43 100. Boyle GM, Roucou X, Nagley P, Devenish RJ, Prescott M 66. Shan XY, Wang LQ, Hoffmaster R, Kruger WD (1999) J Biol (1999) Eur J Biochem 262:315–323 Chem 274:32613–32618 101. Wickman HH, Klein MP, Shirley DA (1964) Phys Rev 67. Robinson KM, Lemire BD (1996) J Biol Chem 271:4061– 152:345–357 4067 102. Mayhew SG (1978) Eur J Biochem 85:535–547 68. Gin P, Hsu AY, Rothman SC, Jonassen T, Lee PT, Tzagoloff A, 103. Lindahl PA, Day EP, Kent TA, Orme-Johnson WH, Mu¨nck E Clarke CF (2003) J Biol Chem 278:25308–25316 (1985) J Biol Chem 260:1160–1173

123 1052 J Biol Inorg Chem (2007) 12:1029–1053

104. Andrew CR, Kemper LJ, Busche TL, Tiwari AM, Kecskes MC, 138. Yu L, Dong J-H, Yu C-A (1986) Biochim Biophys Acta Bio- Stafford JM, Croft LC, Lu S, Moenne-Loccoz P, Huston W, energ 852:203–211 Moir JWB, Eady RR (2005) Biochemistry 44:8664–8672 139. Tegoni M, Silvestrini MC, Guigliarelli B, Asso M, Brunori M, 105. Miyoshi H, Tokutake N, Imaeda Y, Akagi T, Iwamura H (1995) Bertrand P (1998) Biochemistry 37:12761–12771 Biochim Biophys Acta 1229:149–154 140. Anemu¨ller S, Bill E, Scha¨fer G, Trautwein A, Teixeira M (1992) 106. Babcock M, deSilva D, Oaks R, DavisKaplan S, Jiralerspong S, Eur J Biochem 210:133–138 Montermini L, Pandolfo M, Kaplan J (1997) Science 276:1709– 141. Lester RL, Crane FL (1959) J Biol Chem 234:2169–2175 1712 142. Waldeck AR, Stowell MHB, Lee HK, Hung SC, Matsson M, 107. Foury F, Cazzalini O (1997) FEBS Lett 411:373–377 Hederstedt L, Ackrell BAC, Chan SI (1997) J Biol Chem 108. Tangeras A, Flatmark T, Backstrom D, Ehrenberg A (1980) 272:19373–19382 Biochim Biophys Acta 589:162–175 143. Lukoyanov D, Berry SM, Lu Y, Antholine WE, Scholes CP 109. Kispal G, Csere P, Prohl C, Lill R (1999) EMBO J 18:3981– (2002) Biophys J 82:2758–2766 3989 144. Gamelin DR, Randall DW, Hay MT, Houser RP, Mulder TC, 110. Petrat F, de Groot H, Rauen U (2001) Biochem J 356:61–69 Canters GW, de Vries S, Tolman WB, Lu Y, Solomon EI (1998) 111. Petrat F, Weisheit D, Lensen M, de Groot H, Sustmann R, J Am Chem Soc 120:5246–5263 Rauen U (2002) Biochem J 362:137–147 145. Nittis T, George GN, Winge DR (2001) J Biol Chem 112. Sturm B, Bistrich U, Schranzhofer M, Sarsero JP, Rauen U, 276:42520–42526 Scheiber-Mojdehkar B, de Groot H, Ioannou P, Petrat F (2005) 146. Heaton DN, George GN, Garrison G, Winge DR (2001) Bio- J Biol Chem 280:6701–6708 chemistry 40:743–751 113. Lange H, Kispal G, Lill R (1999) J Biol Chem 274:18989– 147. Ludovico P, Sansonetty F, Corte-Real M (2001) Microbiology 18996 147:3335–3343 114. Agar JN, Krebs C, Frazzon J, Huynh BH, Dean DR, Johnson 148. Sickmann A, Reinders J, Wagner Y, Joppich C, Zahedi R, MK (2000) Biochemistry 39:7856–7862 Meyer HE, Schonﬁsch B, Perschil I, Chacinska A, Guiard B et al 115. Franco R, Moura JJG, Moura I, Huynh BH, Forbes WS, Ferreira (2003) Proc Natl Acad Sci USA 100:13207–13212 GC (1995) J Biol Chem 270:26352–26357 149. Huh W-K, Falvo JV, Gerke LC, Carroll AS, Howson RW, 116. Haile DJ, Rouault TA, Harford JB, Kennedy MC, Blondin GA, Weissman JS, O’Shea EK (2003) Nature 425:686–691 Beinert H, Klausner RD (1992) Proc Natl Acad Sci USA 150. Prokisch H, Scharfe C, Camp DG, Xiao WZ, David L, Andreoli 89:11735–11739 C, Monroe ME, Moore RJ, Gritsenko MA, Kozany C, Hixson 117. Henze K, Martin W (2003) Nature 426:127–128 KK, Mottaz HM, Zischka H, Uefﬁng M, Herman ZS, Davis RW, 118. Mu¨hlenhoff U, Lill R (2000) Biochim Biophys Acta Bioenerg Meitinger T, Oefner PJ, Smith RD, Steinmetz LM (2004) PLoS 1459:370–382 Biol 2:795–804 119. Kilpatrick LK, Kennedy MC, Beinert H, Czernuszewicz RC, 151. Forster J, Famili I, Fu P, Palsson BO, Nielsen J (2003) Genome Qiu D, Spiro TG (1994) J Am Chem Soc 116:4053–4061 Res 13(2):244–253 120. Jackson TA, Karapetian A, Miller AF, Brunold TC (2004) J Am 152. Ohlmeier S, Kastaniotis AJ, Hiltunen JK, Bergmann U (2004) Chem Soc 126:12477–12491 J Biol Chem 6:3956–3979 121. Un S, Tabares LC, Cortez N, Hiraoka BY, Yamakura F (2004) 153. Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle A, J Am Chem Soc 126:2720–2726 Dephoure N, O’Shea EK, Weissman JS (2003) Nature 425:737– 122. Srinivasan C, Liba A, Imlay JA, Valentine JS, Gralla EB (2000) 741 J Biol Chem 275:29187–29192 154. Biswas SK, Yamaguchi M, Naoe N, Takashima T, Takeo K 123. Werth MT, Sices H, Cecchini G, Schroder I, Lasage S, Gunsalus (2003) J Electron Microsc 52:133–143 RP, Johnson MK (1992) FEBS Lett 299:1–4 155. Trumpower B (1990) J Biol Chem 265:11409–11412 124. Hirst J, Sucheta A, Ackrell BAC, Armstrong FA (1996) J Am 156. Murphy MEP, Nall BT, Brayer GD (1992) J Mol Biol 227:160– Chem Soc 118:5031–5038 176 125. Sucheta A, Ackrell BAC, Cochran B, Armstrong FA (1992) 157. Daum G et al (1982) J Biol Chem 257:13028–13033 Nature 356:361–362 158. Goodin DB, Davidson MG, Roe JA, Mauk AG, Smith M (1991) 126. Link TA, Hagen WR, Pierik AJ, Assmann C, von Jagow G Biochemistry 30:4953–4962 (1992) Eur J Biochem 208:685–691 159. Capeillereblandin G, Bray RC, Iwatsubo M, Labeyrie F (1975) 127. Beinert H, Shaw RW (1977) Biochim Biophys Acta 462:121–130 Eur J Biochem 54:549–566 128. Aasa R, Albracht SPJ, Falk KE, Lanne B, Vanngard T (1976) 160. Ebert CE, Ghosh M, Wang YD, Beattie DS (2003) Biochim Biochim Biophys Acta 422:260–272 Biophys Acta 1607:65–78 129. Gorbikova EA, Vuorilehto K, Wikstrom M, Verkhovsky MI 161. Orme-Johnson NR, Hansen RE, Beinert H (1971) Biochem (2006) Biochemistry 45:5641–5649 Biophys Res Commun 45:871 130. Nicholls P, Elliott WB (1974) In: Worwood M, Jacobs A (eds) 162. Salerno JC (1984) J Biol Chem 259:2331–2336 Iron in biochemistry and medicine. Academic, New York 163. Svensson-Ek M, Abramson J, Larsson G, Tornroth S, Brzezinski 131. Wittenberg BA, Kampa L, Wittenberg JB, Blumberg WE, P, Iwata S (2002) J Mol Biol 321(2):329–339 Peisach J (1968) J Biol Chem 243:1863–1870 164. Babcock GT, Vickery LE, Palmer G (1978) J Biol Chem 132. Coulson AFW, Erman JE, Yonetani T (1971) J Biol Chem 253:2400–2411 246:917–924 165. Cheesman MR, Oganesyan VS, Watmough NJ, Butler CS, 133. Goodin DB, McRee DE (1993) Biochemistry 32:3313–3324 Thomson AJ (2004) J Am Chem Soc 126:4157–4166 134. Bellei M, Jakopitsch C, Battistuzzi G, Sola M, Obinger C (2006) 166. Beinert H, Ackrell BAC, Kearney EB, Singer TP (1974) Bio- Biochemistry 45:4768–4774 chem Biophys Res Commun 58:564–572 135. Cassanova N, O’Brien KM, Stahl BT, McClure T, Poyton RO 167. Peterson J, Vibat C, Gennis RB (1994) FEBS Lett 355:155–156 (2005) J Biol Chem 280:7645–7653 168. Svensson B, Andersson KK, Hederstedt L (1996) Eur J Biochem 136. Reynolds MF, Shelver D, Kerby RL, Parks RB, Roberts GP, 238:287–295 Burstyn JN (1998) J Am Chem Soc 120:9080–9081 169. Jonassen T, Proft M, Randez-Gil F, Schultz JR, Marbois BN, 137. Lett CM, Guillemette JG (2002) Biochem J 362:281–287 Entian KD, Clarke CF (1998) J Biol Chem 273:3351–3357

123 J Biol Inorg Chem (2007) 12:1029–1053 1053

170. Rea S (2001) FEBS Lett 509:389–394 180. Hoja U, Marthol S, Hofmann J, Stegner S, Schulz R, Meier S, 171. Liu KE, Lippard SJ (1991) J Biol Chem 266:12836–12839 Greiner E, Schweizer E (2004) J Biol Chem 279:21779–21786 172. Fox BG, Hendrich MP, Surerus KK, Anderson KK, Froland 181. Ollagnier-de Choudens S, Fontecave M (1999) FEBS Lett WA, Lipscomb JD, Mu¨nck E (1993) J Am Chem Soc 115:3688– 453:25–28 3701 182. Cosper MM, Jameson GNL, Hernandez HL, Krebs C, Huynh 173. Regev-Rudzki N, Karniely S, Ben-Haim NN, Pines O (2005) BH, Johnson MK (2004) Biochemistry 43:2007–2021 Mol Biol Cell 16:4163–4171 183. Branda SS, Cavadini P, Adamec J, Kalousek F, Taroni F, Isaya 174. Tong JJ, Feinberg BA (1994) J Biol Chem 269:24920–24927 G (1999) J Biol Chem 274:22763–22769 175. Smith AD, Jameson GNL, Dos Santos PC, Agar JN, Naik S, 184. He YN, Alam SL, Proteasa SV, Zhang Y, Lesuisse E, Dancis A, Krebs C, Frazzon J, Dean DR, Huynh BH, Johnson MK (2005) Stemmler TL (2004) Biochemistry 43:16254–16262 Biochemistry 44:12955–12969 185. Bou-Abdallah F, Adinolﬁ S, Pastore A, Laue TM, Chasteen ND 176. Pelzer W, Muhlenhoff U, Diekert K, Siegmund K, Kispal G, Lill (2004) J Mol Biol 341:605–615 R (2000) FEBS Lett 476:134–139 186. Dawson JH, Bracete AM, Huff AM, Kadkhodayan S, Zeitler 177. Picciocchi A, Douce R, Alban C (2003) J Biol Chem CM, Sono M, Chang CK, Loewen PC (1991) FEBS Lett 278:24966–24975 295:123–126 178. Ugulava NB, Gibney BR, Jarrett JT (2001) Biochemistry 187. Ioannidis N, Cooper CE, Poole RK (1992) Biochem J 288:649– 40:8343–8351 655 179. Duin EC, Lafferty ME, Crouse BR, Allen RM, Sanyal I, Flint 188. Macheroux P, Hill S, Austin S, Eydmann T, Jones T, Kim SO, DH, Johnson MK (1997) Biochemistry 36:11811–11820 Poole R, Dixon R (1998) Biochem J 332:413–419

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