ORIGINAL PAPER

Electron paramagnetic resonance and Mossbauer spectroscopyof intact mitochondria from respiring Saccharomyces cerevisiae

Brandon N. Hudder Æ Jessica Garber Morales ÆAudria Stubna Æ Eckard Munck ÆMichael 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

under anaerobic conditions. Microscopic images were lar-

gely devoid of contaminants, and samples consumed O2 in

an NADH-dependent manner. Protein and metal concen-

trations of packed mitochondria were determined, as was the

percentage of external void volume. Samples were similarly

packed into electron paramagnetic resonance tubes, either in

the as-isolated state or after exposure to various reagents.

Analyses revealed two signals originating from species that

could be removed by chelation, including rhombic Fe3+

(g = 4.3) and aqueous Mn2+ ions (g = 2.00 with Mn-based

hyperfine). Three S = 5/2 signals from Fe3+ hemes were

observed, probably arising from cytochrome c peroxidase

and the a3:Cub site of cytochrome c oxidase. Three Fe/S-

based signals were observed, with averaged g values of 1.94,

1.90 and 2.01. These probably arise, respectively, from the

[Fe2S2]+ cluster of succinate dehydrogenase, the [Fe2S2]+

cluster of the Rieske protein of cytochrome bc1, and the

[Fe3S4]+ cluster of aconitase, homoaconitase or succinate

dehydrogenase. Also observed was a low-intensity isotropic

g = 2.00 signal arising from organic-based radicals, and a

broad signal with gave = 2.02. Mossbauer spectra of intact

mitochondria were dominated by signals from Fe4S4 clusters

(60–85% of Fe). The major feature in as-isolated samples,

and in samples treated with ethylenebis(oxyethylenenitril-

o)tetraacetic acid, dithionite or O2, was a quadrupole doublet

with DEQ = 1.15 mm/s and d = 0.45 mm/s, assigned to

[Fe4S4]2+ clusters. Substantial high-spin non-heme Fe2+

(up to 20%) and Fe3+ (up to 15%) species were observed.

The distribution of Fe was qualitatively similar to that

suggested by the mitochondrial proteome.

Keywords Iron � Sulfur � Cluster assembly �Heme biosynthesis � Non-heme

Abbreviations

CoQ Coenzyme Q

DTT Dithiothreitol

EDTA Ethylenediaminetetraacetic acid

EGTA Ethylenebis(oxyethylenenitrilo)

tetraacetic acid

EPR Electron paramagnetic resonance

ETF Electron transfer flavoprotein

HEPES N-(2-Hydroxyethyl)piperazine-N0-ethanesulfonic acid

IM Inner membrane

IMS Intermembrane space

NHE Normal hydrogen electrode

OM Outer membrane

SH buffer 0.6 M sorbitol/20 mM N-(2-hydroxyethyl)

piperazine-N0-ethanesulfonic acid buffer pH

7.4

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00775-007-0275-1) contains supplementarymaterial, which is available to authorized users.

B. N. Hudder � J. G. Morales � P. A. Lindahl (&)

Department of Chemistry,

Texas A&M University,

College Station, TX 77843-3255, USA

e-mail: lindahl@mail.chem.tamu.edu

A. Stubna � E. Munck � M. P. Hendrich

Department of Chemistry,

Carnegie Mellon University,

Pittsburgh, PA 15213-2683, USA

P. A. Lindahl

Department of Biochemistry and Biophysics,

Texas A&M University,

College Station, TX 77843, USA

123

J Biol Inorg Chem (2007) 12:1029–1053

DOI 10.1007/s00775-007-0275-1

SP buffer 1.2 M sorbitol/20 mM potassium

phosphate buffer pH 7.4

Introduction

Mitochondria are the cellular organelles in which oxidative

phosphorylation and a myriad of related processes

involving iron, copper and manganese occur. These bran-

ched tubular structures have an outer membrane (OM), an

aqueous intermembrane space (IMS), an inner membrane

(IM) and an aqueous matrix region. The IM is highly

invaginated, with cristae protruding into the aqueous

matrix region. Imported iron ions are used in heme and

iron–sulfur (Fe/S) cluster biosynthesis. A portion of these

nascent prosthetic groups are incorporated into mitochon-

drial apoproteins, while the remainder are exported to the

cytosol. Imported copper and manganese ions are installed

into cytochrome c oxidase and manganese superoxide

dismutase, respectively.

The proteins involved in these processes can be cate-

gorized in terms of the metal centers they contain. Proteins

containing Fe2S2, Fe3S4 and/or Fe4S4 clusters include

succinate dehydrogenase [1–3], the Rieske protein [4],

aconitase and homoaconitase [5, 6], ferredoxin/adreno-

doxin [7–10], biotin synthase [11–15] and lipoic acid

synthase [16–18]. Dihydroxyacid dehydratase catalyzes the

dehydration of an intermediate in the biosynthesis pathway

of branched-chain amino acids [19]. Although the metal

center in this enzyme has not been well studied, the

homologous enzyme from Escherichia coli contains an

Fe4S4 cluster [20]. Such clusters are also found in scaffold

proteins which are used in the synthesis of Fe/S clusters,

including Isu1p, Isu2p, Isa1p and Nfu1p [21–24]. A

BLAST search suggests that the open reading frame

YOR356W encodes the flavin adenine dinucleotide-con-

taining and Fe4S4-containing electron transfer flavoprotein

(ETF) dehydrogenase [25–27].

Other mitochondrial proteins contain heme groups.

Heme b is found in cytochrome bc1 [28], cytochrome c

peroxidase [29], succinate dehydrogenase and flavocyto-

chrome b2 [30]. Heme a is found in cytochrome c oxi-

dase [31], while heme c is contained in cytochrome c1

and in both isoforms of cytochrome c [32]. Heme

monooxygenase catalyzes the conversion of heme b to

heme a within the heme biosynthetic pathway [33]. The

homolog from E. coli contains heme b and heme a

prosthetic groups [34–36]. When yeast cells are grown

under respiratory conditions, the heme-b-containing cat-

alase A (Cta1p) is targeted to the mitochondrial matrix

[37].

Another group of proteins are involved in iron traffick-

ing. Isa2p [38] and Yfh1p [39] help import Fe2+ ions into

the matrix and insert iron into ferrochelatase (Hem15p) for

heme biosynthesis [40–42] and into scaffold proteins for

Fe/S synthesis. IM proteins Mrs3p, Mrs4p, Mmt1p and

Mmt2p carry iron into mitochondria [43, 44]. Heme O

synthase (Cox10p) and heme A synthase (Cox15p) bind

intermediate states of hemes [33, 35, 45]. Cytochrome c

heme lyase (Cyc3p) and cytochrome c1 heme lyase (Cyt2p)

install heme c into cytochromes c and c1, respectively [46].

Mdl1p exports heme groups [47], while Atm1p and Erv1p

export Fe/S clusters [48, 49]. Coq7p is a yeast mitochon-

drial protein that contains a diiron center and serves as a

monooxygenase/hydroxylase in coenzyme Q (CoQ) bio-

synthesis [50, 51].

Cytochrome c oxidase is the best-known copper-con-

taining mitochondrial protein. The Cox1p subunit of this

complex contains one copper ion (CuB) adjacent to heme

a3 in its active site, while the electron-transfer CuA site in

Cox2p contains two copper ions [52]. Cox23p, Cox17p,

Sco1p and Cox11p are chaperones that import Cu ions into

mitochondria and insert them into Cox1p and Cox2p dur-

ing their assembly [53–55]. Copper ions in these chaper-

ones are in the diamagnetic Cu+ oxidation state. A small

amount of the cytosolic copper-containing (Cu–Zn)

superoxide dismutase (Sod1p) appears to localize in the

IMS of mitochondria [56]. Approximately 90% of mito-

chondrial copper is found in the matrix as a nonproteina-

ceously bound pool of Cu+ ions [57].

Manganese superoxide dismutase (Sod2p) appears to be

the only manganese-containing enzyme in Saccharomyces

cerevisiae mitochondria. The mitochondrial manganese

chaperone protein (Mtm1p) helps to import manganese

ions and to install one of these ions into matrix-localized

apo-Sod2p [58].

Flavins and ubiquinone can be stabilized in an S = 1/2

semiquinone state that affords electron paramagnetic res-

onance (EPR) signals at the free-electron g value, 2.00.

Flavin-containing mitochondrial proteins include a-keto-

glutarate dehydrogenase [59], D-lactate cytochrome c ox-

idoreductases [60], glutathione reductase [61], thioredoxin

reductase [62], glycerol-3-phosphate dehydrogenase [63],

D-arabinono-1,4-lactone oxidase [64], acetolactate synthase

[65], methylene tetrahydrofolate reductase [66], succinate

dehydrogenase [67], Coq6p [68], Mmf1p [69] and ETF

dehydrogenase [25–27].

The most important spectroscopic technique that has

been applied to intact mitochondria is EPR, dating from the

pioneering work of Beinert [70], who initially described

high-spin heme signals from cytochrome c oxidase [71].

The gave = 2.01 EPR signal from the inactivated [Fe3S4]+

form of aconitase was observed in crude intact rat-heart

mitochondria exposed to H2O2 [72]. EPR signals from the

1030 J Biol Inorg Chem (2007) 12:1029–1053

123

Rieske cluster of cytochrome bc1 and the [Fe2S2]+ cluster

of succinate dehydrogenase have also been observed in

intact mitochondria [73–78]. EPR spectra of intact mito-

chondria were examined to determine the effect of abol-

ishing heme biosynthesis on succinate dehydrogenase and

the Rieske protein [75] and to determine the effects of Ca2+

and Mn2+ ions [79, 80]. Adrenodoxin levels in intact

human placental mitochondria were examined by EPR

[81]. Respiratory complexes in submitochondrial fractions

have also been examined [82–84]. In contrast, there has

been just one report of a Mossbauer spectrum of intact

mitochondria, specifically of a strain in which yfh1 was

deleted [39]. This genetic modification causes iron to

accumulate in the matrix, and the observed Mossbauer

spectral intensity exclusively reflected the accumulated

iron. The ‘‘control’’ Mossbauer spectrum of wild-type

mitochondria was devoid of any signals.

This overview highlights the complexity of transition

metal metabolism occurring within these organelles. We

report on our efforts to establish a few simple yet unes-

tablished aspects of iron metabolism in yeast mitochondria,

namely, the absolute concentration of iron and of overall

protein contained therein, and the proportion of that iron

present in various types of centers (e.g., hemes, Fe/S

clusters, etc.). Our approach was to investigate mitochon-

dria from S. cerevisiae using EPR and Mossbauer spec-

troscopy along with various bioanalytical characterizations.

For the first time using whole mitochondria, the absolute

spin concentrations of detectable metal protein species

have been quantified from EPR spectra. We investigated

intact mitochondria prepared under different redox and/or

isolation conditions. We determined the proportion of

excluded buffer in these packed samples, which, when

combined with metal and protein determinations of the

packed samples, allowed us to estimate the absolute iron

concentration contained within these organelles. This

information, when combined with our spectroscopic

results, allowed us to estimate, albeit in broad terms, how

iron is distributed within the organelle. This distribution

was then compared with that calculated from the iron-

containing proteins known to be present in the mitochon-

drial proteome.

Materials and methods

Cell growth and isolation of mitochrondia

S. cerevisiae cells (strain D273-10B) were grown on SSlac

medium (0.3% glucose, 1.7% lactate) [72 g yeast extract,

25 g ammonium chloride, 25 g potassium hydrogen phos-

phate, 12.5 g NaCl, 12.5 g CaCl2, 14.4 g MgCl2, 12.5 g

glucose and 0.7 L of 60% sodium lacate syrup (Fisher) in

25-L solution] in a custom-built thermostatically controlled

autoclavable 25-L glass fermenter in which cultures were

stirred and bubbled with pure O2 at a rate of approximately

3 L/min, dispersed through a fine glass frit with a diameter

of 5 cm. Under these growth conditions, cells ferment on

glucose at early stages of growth and then switch to res-

piration on lactate once the glucose has been consumed.

Harvesting commenced when the optical density of a 1-cm

solution at 600 nm reached 1.2–1.4. The culture was

chilled to 278 K and harvested at 5,000 rpm using a

Sorvall SLC-6000 rotor and a Sorvall Evolution centrifuge.

Immediately after harvesting and without freezing the

pelleted cells, mitochondria were isolated essentially as

described in [85] except that all steps were performed

anaerobically. Cell paste (100–150 g) was transferred into

a refrigerated argon-atmosphere glove box (M. Braun)

maintained at approximately 278 K and approximately

1 ppm O2 as monitored continuously using a model 310

Teledyne analyzer. Buffers used in the isolation were

degassed on a Schlenk line. For some preparations, all

isolation buffers were supplemented with ethylenedi-

aminetetraacetic acid (EDTA) or ethylenebis(oxyethy-

lenenitrilo)tetraacetic acid (EGTA) (Acros) at final

concentrations of 1 or 10 mM. In other preparations, no

metal chelators were included. Cell paste was suspended

in a 100 mM tris(hydroxymethyl)aminomethane sulfate/

10 mM dithiothreitol (DTT) buffer (500 mL) and then

spun at 5,000 rpm for 5 min in the SLC-6000 rotor. Sub-

sequent centrifugations were performed under these con-

ditions unless otherwise stated. The resulting pellet was

suspended in 1.2 M sorbitol/20 mM potassium phosphate

buffer, pH 7.4 (500 mL), hereafter referred to as SP buffer,

using a rubber policeman. The resulting suspension was

centrifuged, resuspended in SP buffer (500 mL), centri-

fuged again, and resuspended again in the same buffer. Cell

walls were disrupted by adding 3 mg of 100 units/mg yeast

lytic enzyme (Sigma) per gram of cell paste. The resulting

spheroplasts were centrifuged, suspended in SP buffer

(500 mL) and then centrifuged. The pellet was resuspended

in 250 mL of 1.2 M sorbitol/40 mM N-(2-hydroxyethyl)

piperazine-N0-ethanesulfonic acid (HEPES) pH 7.4 and

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)

during a period of 2–4 min. The suspension was centri-

fuged at 2,500 rpm for 5 min, and the supernatant was

transferred to a fresh centrifuge bottle and centrifuged

again under the same conditions. The supernatant, which

consisted of crude mitochondria, was then centrifuged at

10,000 rpm in a Sorvall SLA-1500 rotor for 10 min. The

resulting pellet was resuspended in 200 mL of a 0.6 M

sorbitol/20 mM HEPES buffer pH 7.4, hereafter referred to

as SH buffer. The resulting solution was centrifuged three

J Biol Inorg Chem (2007) 12:1029–1053 1031

123

more times, in the manner described in the previous three

sentences, and the final pellet of crude mitochondria was

resuspended in 20 mL SH buffer. This solution was loaded

onto a discontinuous gradient solution composed of 10 mL

of 15% and 10 mL of 20% (w/v) Histodenz1 (Sigma)

prepared in SH buffer and contained in Beckman Ultra

ClearTM centrifuge tubes. The tubes were placed in the

buckets of an SW-32Ti rotor (Beckman Coulter). The

buckets were sealed, removed from the box and spun at

9,000 rpm in an SW-32Ti rotor (Beckman Coulter) for

1.5 h using a Beckman L7 ultracentrifuge. The buckets

were returned to the box, and the tubes were placed in a

support which allowed the pure mitochondrial band at the

interface of the gradient to be collected after first removing

the layer above the band. From 150 g cell paste, a total of

5–15 mL of mitochondrial solution in the ‘‘as-isolated’’

state was obtained using three to six buckets depending on

the yield. The only reductant used during the procedure

was DTT and then only at an early step of the isolation

procedure before cell walls were disrupted. E�0 for the

disulfide/DTT half-cell is �330 mV versus the normal

hydrogen electrode (NHE) [86]. Anaerobically prepared

isolation buffers undoubtedly contained a trace of oxidiz-

ing ability [87]. Both factors considered, the resulting

solution potential of mitochondria in the non-redox-buf-

fered ‘‘as-isolated’’ state was estimated to be between �0.1

and 0 mV versus NHE. Prior to freezing, some samples

were exposed to air (typically for 1 day at 277 K), sodium

dithionite (10 mM at pH 7.5 or 8.5), potassium ferricyanide

(1 mM) or nitric oxide (1 atm).

For Mossbauer spectroscopy studies, S. cerevisiae cells

were grown similarly except that the medium was sup-

plemented with 20 lM 57FeCl3. With use of a custom-

made DelrinTM insert that fit in the buckets of the SW-32Ti

rotor, isolated mitochondria were packed tightly into

Mossbauer cuvettes by centrifugation, typically at

9,000 rpm for 2 h. Samples were then frozen inside the

glove box by contact with a liquid-nitrogen-cooled alumi-

num block. There was some variation in speed and duration

used in packing, resulting in some differences in terms of

observed 57Fe concentrations. Each spectrum presented

here was recorded with an approximately 40 mCi 57Co

source.

Electron and fluorescence microscopy

One milliliter of as-isolated mitochondrial solution was

microcentrifuged (Fisher Scientific) at 6,400 rpm for 5 min

in a 1.5-mL Eppendorf tube. The pellet was resuspended in

SH buffer and glutaraldehyde (2.0% v/v final concentra-

tion) was added. The solution was recentrifuged and the

pellet was resuspended in 1% osmium tetroxide and 0.5%

potassium ferrocyanide (w/v) in SH buffer. This was fol-

lowed by en bloc staining using 1% uranyl acetate in SH

buffer. Samples were dehydrated by incubation in

increasingly concentrated ethanol solutions and then

embedded using epoxy-based resin. Thin-sectioning was

performed using a glass knife/water trough on a micro-

tome, followed by retrieval of the thin sections using 200

mesh grids. Positive staining of these sections was per-

formed using lead acetate/sodium hydroxide [88]. Images

were obtained using a JEOL 1200 EX transmission electron

microscope.

For fluorescence images, equivalent mitochondrial solu-

tions were incubated in SH buffer, containing 500 nM

MitoTracker1 (Molecular Probes) or, in another experi-

ment, 1 lM ERTracker1 at 310 K for 45 min. The solution

was centrifuged, and the pellet was resuspended in SH buf-

fer. Images were obtained using a Bio-Rad Radiance

2000 MP instrument equipped with a ·63 (water-immer-

sion) objective.

Oxygen consumption measurements

A sample of non-chelator-treated intact mitochondria was

suspended in SH buffer. A 5-mL portion was assayed for

protein concentration using the biuret method [89] as

described in ‘‘Protein and metal ion concentrations.’’

Another portion of the intact mitochondrial solution was

divided into three samples. One sample was incubated

anaerobically for 4–5 h with 10 mM EDTA, another was

incubated similarly with 10 mM EGTA, and the remaining

sample was not treated. Each sample (1.2 mL) was injected

into 29 ± 1 mL of air-saturated SH buffer containing

1.5 mM NADH, 0.2 mM ADP, 2 mM MgCl2, 20 mM

phosphates pH 7.4, 250 mM sucrose and 10 mM KCl,

essentially as described in [90]. The solution was main-

tained at 298 K in a water-jacketed glass vessel which

contained no gas head space. Included in this vessel was a

Clark oxygen electrode (YSI Bioanalytical Products). The

final protein concentration was 0.10 mg/mL.

Electron paramagnetic resonance

Custom-built DelrinTM inserts were designed to fit within

the buckets of the SW-32Ti rotor. Holes were drilled into

the center of these inserts, with a diameter just sufficient to

fit a modified EPR tube (4.96-mm outer diameter; 3.39-mm

inner diameter; 80-mm long; Wilmad/Lab Glass, Buena,

NJ, USA). A 2-mm-long cylinder of silicone rubber was

inserted at the bottom of the hole. The brown mitochon-

drial solution obtained from the gradient step described in

‘‘Cell growth and isolation of mitochondria’’ was diluted

1032 J Biol Inorg Chem (2007) 12:1029–1053

123

with an equal volume of SH buffer. Tubes were filled with

this solution and the entire assembly was sealed, removed

from the box and spun by centrifugation at 9,000 rpm for

1 h. Samples were returned to the box, and the supernatant

was replaced with additional mitochondrial solution. This

process was repeated until the volume of tightly packed

mitochondria at the bottom of the tube reached approxi-

mately 400 lL. EPR tubes were removed from the inserts

and frozen in less than 1 min using liquid N2. Two to four

EPR samples were prepared from a solution of gradient-

purified mitochondria isolated from 25 L of culture.

One end of a stainless steel wire (20 cm · 0.5-mm

diameter) was attached to one end of a stainless steel rod

(20 cm · 4.8-mm diameter), with the wire extended

coaxially with the rod. Approximately 5 cm beyond the

point of attachment, the wire was bent back towards the rod

(like a hairpin) and coiled around itself up towards the rod.

The outer diameter of the coil at the base of the hairpin was

slightly less than the inner diameter of the modified EPR

tube, while the outer diameter of the remainder of the coil

was slightly greater than the inner diameter of the EPR

tube. In this way, the wire coil fit snugly into the upper

region of the EPR tubes. The entire assembly was just

sufficiently robust to be inserted into and removed from the

EPR cavity. Spectra were obtained with a Bruker EMX X-

band EPR spectrometer operating in perpendicular mode

with an Oxford Instruments EM910 cryostat. Signals were

simulated with SpinCount written by one of the authors

(M.P.H.). Signal intensities were quantified relative to a

CuEDTA spin standard using the same software.

Protein and metal ion concentrations

A line was drawn on the exterior of the EPR tubes to

indicate the height of the packed mitochondria. The

organelles were thawed and quantitatively transferred to

plastic screw-top vials using a slightly twisted quartz rod

and a minimal volume of SH buffer. The volume of packed

organelles was determined by weighing the tubes before

and after filling them with an equivalent volume of water,

and then dividing the difference by the density of water.

The final volume of the solution in the screw-top vial,

typically 5 mL, was similarly determined. The ratio of

these two volumes constituted the dilution factor by which

measured protein and metal concentrations, obtained using

the solution in the vial, were multiplied to yield the

respective concentrations in packed mitochondria.

Samples contained in the vial were sonicated using a

Branson Sonifier 450 operating for 5–10 min at 60%

capacity. Protein analyses were performed in either of two

ways, namely, by quantitative amino acid analysis, which

is the most accurate method available [91], or by the biuret

colorimetric method. Relative to amino acid analysis, the

results obtained using the biuret method were similar

within the uncertainty of the measurements. For quantita-

tive amino acid analysis, aliquots were hydrolyzed in 6 M

HCl/2% phenol at 383 K and analyzed using a Hewlett-

Packard AminoQuant system. Amino acid percentages

were similar among preparations. Primary and secondary

amino acids present in the samples were derivatized

using o-phthalaldehyde and 9-fluoromethylchloroformate,

respectively. Metal concentrations were determined by

atomic absorption spectrometry (PE AAnalyst 700 oper-

ating in furnace mode) and by inductively coupled plasma

mass spectrometry (PerkinElmer). Sonicated samples

(250–400 lL) were digested using an equal volume of

15.8 M trace-metal-grade HNO3 (Fischer Scientific) in a

sealed plastic tube that was then incubated overnight at

353 K. The resulting solution was diluted with deionized

and distilled H2O to a final HNO3 concentration of 0.2 M.

Percentage of external solution in packed samples

Custom-built Lexan ‘‘graduated cylinders’’ were con-

structed within inserts that fit within buckets of the SW-

32Ti rotor (Fig. 1). These inserts were used to accurately

measure the volume of a packed mitochondria sample,

obtained by loading a solution of isolated mitochondria and

spinning the sample for 1 h at 9,000 rpm (10,000g). The

supernatant was decanted and the volume of the packed

sample was measured using this apparatus. This volume

(Vpel) was assumed to be composed of the volume of

the mitochondria plus the volume of excluded water:

Fig. 1 Graduated cylinder used to measure the volume of packed

mitochondria samples

J Biol Inorg Chem (2007) 12:1029–1053 1033

123

(Vpel = Vmito + VH2O). To determine the ratio VH2O/Vpel, a

1.00-mL stock solution of radioactively labeled sucrose

(American Radiolabeled Chemicals, 625 mCi/mmol), pre-

pared in SH buffer (with Cstock* in counts per minute per

milliliter given in Table 1 for each experiment), was added

to the pellet and the pellet was resuspended. The inserts

were spun by centrifugation as described above, the

supernatant was removed, the volume (Vsup1) was deter-

mined using a gastight syringe (Hamilton), and the con-

centration of radioactivity (Cstock* ) was determined by

scintillation counting (Beckman 5000SL). Assuming that

none of the sucrose entered the mitochondria, the excluded

water will also have a concentration of radioactivity given

by Cstock* . The conservation of matter suggests that

C�stockVstock ¼ C�sup1Vsup1 þ C�sup1VH2O1:

This equation was solved for VH2O1. The resulting pellet

was found to have essentially the same volume as the

original pellet. This pellet, containing radioactively labeled

sucrose in the external volume, was resuspended with a

solution of nonradioactively labeled sucrose, and the other

steps of the same process were repeated. In this case, the

resulting concentration of radioactivity in the supernatant

fraction was called Csup2* and the corresponding

conservation of matter relationship becomes

C�sup1 � VH2O2¼ C�sup2 � Vsup2 þ C�sup2 � VH2O2

:

This equation was solved for VH2O2. The average of the two

values for VH2O was divided by Vpel, affording the fraction

of the pellet volume due to excluded water.

Results

Characterization of intact mitochondria

Intact yeast mitochondria were isolated as described in

‘‘Materials and methods.’’ Some preparations were isolated

without adding a metal chelator to the isolation buffers,

while others were isolated in the presence of either EDTA

or EGTA. These chelators were added to remove adven-

titious metal ions associated with mitochondria. EGTA is

unable to penetrate biological membranes [92], while this

property is uncertain with respect to EDTA. However,

EDTA has been used in isolating mitochondria [93] and as

far as we are aware, there have been no reports of EDTA

stripping essential metal ions from these organelles.

We assayed a number of preparations for purity and

membrane integrity using electron microscopy. Although

significant size dispersion was typically evident (Fig. 2,

top), there was no obvious evidence of impurities (bacteria

or Golgi apparatus) or disrupted membrane structures.

Sample morphology was independent of the method of

isolation (as-isolated, EDTA-treated or EGTA-treated).

Our images are similar to those obtained in the classical

studies of Hackenbrock [94] and more recently [95]. Dis-

persion probably results from the dynamic fission and

fusion processes that occur in yeast mitochondria [96].

Confocal microscopic images reveal that mitochondria

form extensive tubelike networks extending throughout the

cell [97]. These dynamic changes in size and shape would

appear to render the concept of the number of mitochondria

per cell rather meaningless. A more quantifiable parameter

is the volume occupied by these organelles, and we will use

this parameter throughout this paper.

Fluorescence microscopy was also used to assess purity.

One sample was stained for fluorescence with MitoTrac-

ker1, while another was stained with ERTracker1. The

former dye associates with mitochondria, while the latter

associates with the endoplasmic reticulum. As shown in

Fig. 2, the vast majority of objects in our samples assimi-

lated the MitoTracker1 stain. There was no obvious sign of

endoplasmic reticulum contamination using ERTracker1

(data not shown). Both results suggest that the samples

examined here were relatively pure and intact.

We assayed a number of preparations for their ability to

consume O2. As shown in Fig. 3, preparations incubated in

the absence of chelator or in the presence of EDTA or EGTA

consumed 240, 160 and 200 nmol O2 per minute per

Table 1 Determination of excluded buffer in packed mitochondria samples

Cstock*

(cpm/mL)

Vstock

(mL)

Csup1*

(cpm/mL)

Vsup1

(mL)

VH2O1

(mL)

Csup2*

(cpm/mL)

Vsup2

(mL)

VH2O2

(mL)

Vpel

(mL)

Average

% H2O In Vpel

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

1034 J Biol Inorg Chem (2007) 12:1029–1053

123

milligram of protein, respectively (estimated relative error

of ±20%) when incubated in buffer containing NADH.

Control samples assayed in the absence of NADH consumed

little O2. These rates are similar to those reported previously

[98–100]. We also evaluated the coupling ratio of our

preparations, defined as the rate of O2 consumption with

ADP added to the assay solution divided by the rate of

consumption when ADP was absent. In our fresh samples,

this ratio was approximately 2, similar to previously reported

ratios [99, 100], whereas it approached 1 for mitochondria

stored anaerobically at 278 K for approximately 3 days.

Preparations used for EPR and Mossbauer analyses were

frozen between 6 h and 3 days after they were isolated. We

have not yet been able to correlate the age of the mito-

chondria to specific spectral changes, but we suspect that

spectral features might become slightly broader with age.

We determined protein and metal concentrations in our

packed samples. The mean protein concentration (n = 15)

was 55 ± 13 mg/mL, independent of whether chelators

were or were not included in buffers during mitochondria

isolations. In the absence of chelators, mean Fe, Cu, Mn

and Zn concentrations in our packed mitochondria were

860 ± 480 lM (n = 5), 240 ± 150 lM (n = 5), 40 ±

30 lM (n = 5) and 1,000 ± 200 lM (n = 2), respectively.

Corresponding metal concentrations for packed mitochon-

dria samples isolated in the presence of chelators were

570 ± 100 lM Fe (n = 11), 220 ± 150 lM Cu (n = 11),

20 ± 10 lM Mn (n = 11) and 330 ± 170 lM Zn (n = 5).

The scatter in the Cu, Mn and Zn data precludes us from

drawing strong conclusions regarding the concentration of

these ions in mitochondria. However, the modest scatter for

the protein and Fe concentrations measured in samples

isolated in the presence of chelators indicates that these

concentrations (and their ratio, approximately 10 nmol

Fe/mg protein) are reliable within a relative uncertainty of

25%.

Next, we determined the proportion of the packed

mitochondria samples due to the mitochondria themselves

(rather than to excluded solution). Using the procedure

described in ‘‘Materials and methods,’’ we found the per-

centage of mitochondria in our packed samples to be

77 ± 7 (n = 7), as shown in Table 1. The absolute con-

centrations of protein and Fe concentrations contained in

‘‘neat’’ mitochondria (devoid of solvent) could then be

calculated by dividing the measured concentrations for the

packed samples by 0.77, affording a protein concentration

of approximately 70 mg/mL and Fe concentrations of 0.74

and 1.1 mM for samples isolated in the presence and

absence of chelators, respectively. Given the uncertainty as

Fig. 2 Electron microscopy (top) and fluorescence microscopy

(bottom) images of whole mitochondria isolated from Saccharomycescerevisiae

0 2 4 6 8 10 12 14 16 18 20

0

50

100

150

200

250

300

µnegyx

O M

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’’

J Biol Inorg Chem (2007) 12:1029–1053 1035

123

to whether the Fe removed by chelators had any functional

relevance, and on the basis of 57Fe concentration estimates

based on Mossbauer intensities (see ‘‘Mossbauer spectra of

mitochondria’’), we conclude that the concentration of Fe

in respiring yeast mitochondria is 800 ± 200 lM.

EPR of mitochondria

Mitochondria prepared in three different redox states,

including as-isolated, oxidized and reduced, were packed

tightly into custom-designed EPR tubes so as to expel

external buffer and maximize the intensity of mitochon-

drial EPR signals. As-isolated samples are defined as those

prepared anerobically in the absence of either oxidant or

reductant. Oxidized samples were treated with either O2 or

ferricyanide. Reduced samples were treated with sodium

dithionite. Some samples were prepared in the presence of

the metal chelators EDTA and EGTA, while others were

prepared in the absence of such chelators. This was done in

an attempt to distinguish EPR signals originating from

functional species within mitochondria from species that

were adventitiously bound to the organelle. Owing to

concern that membrane integrity would be compromised

by freeze/thaw cycles, samples were never used twice (i.e.,

they were not thawed, treated in some manner, refrozen

and reanalyzed spectroscopically). Once thawed, samples

were used for protein and metal analyses and then dis-

carded. This procedure produced reasonable but not perfect

correlation between the redox state in which the sample

was prepared and the types and intensities of EPR signals

observed.

EPR signals observed during this study are shown in

Figs. 4 and 5. The principal g values for these signals and

associated spin concentrations are compiled in Table 2.

EPR signals of high-spin (S = 5/2) Fe3+ species were

analyzed with the conventional spin Hamiltonian

H ¼ D½S2z � 35=12þ E=DðS2

x � S2yÞ� þ g0bS � B:

For bB � |D| it is customary to describe the magnetic

properties of the three Kramers doublets by effective

g values, which are dependent on the rhombicity parameter

E/D and the intrinsic g value g0 & 2.0 [101].

Mitochondria prepared in the absence of metal chelators

sometimes exhibited a signal at g = 4.3, as shown in Fig. 4,

spectrum A. This signal is typical of non-heme Fe3+ spe-

cies with E/D & 0.27–0.33. Such spectra also typically

include features at g = 6.9 and 5.0, indicative of a high-

spin Fe3+ heme with E/D = 0.041. The minor signal at

g = 6.0 indicates a second high-spin Fe3+ heme with

E/D & 0 (discussed later). In such samples, the g = 2

region (Fig. 5, spectrum A) is often dominated by a signal

with a six-line hyperfine pattern (magnetic hyperfine cou-

pling constant, a = 90 G) typical of an S = 5/2 Mn(II)

species. A feature at g * 1.94 is also evident but is

obscured by overlap with the Mn(II) signal. In all samples,

the spectral region between g = 4.3 and 2.2 was devoid of

signals. A more recently prepared sample, as-isolated in the

absence of chelators, exhibited a g = 4.3 signal with sig-

nificantly lower intensities than that in Fig. 4, spectrum A

and did not show the Mn(II) signal of Fig. 5, spectrum A;

rather it exhibited the spectrum shown in Fig. 5, spec-

trum B (discussed later). The g & 4.3 and Mn(II) signals

were also either absent or present at low intensity in spectra

of samples as-isolated with chelators included in all iso-

lation buffers. This suggests that all or most of the species

yielding these signals arise from adventitious Mn2+ and

Fe3+ ions that can be chelated by EDTA and EGTA.

80 100 120 140 160 180 200

8 7 6 5 4

B (mT)

A

B

C

D

E

6.9

g

5.0

6.4

5.4

6.0

Fig. 4 Low-field X-band electron paramagnetic resonance (EPR)

spectra of intact mitochondria. A Non-chelator-treated, as-isolated, BEGTA-treated, O2-exposed, C EGTA-treated, as-isolated, D EGTA-

treated, reduced with 10 mM dithionite pH 7.4, E same as D but at pH

8.5. EPR conditions as follows: average microwave frequency,

9.45 GHz; microwave power, 20 mW; modulation amplitude, 10 G;

receiver gain 1 · 104 for A–C, 5.02 · 104 for D and E. Temperature,

10 K. The intensities of D and E have been multiplied by 5 and 2,

respectively

1036 J Biol Inorg Chem (2007) 12:1029–1053

123

The same two high-spin heme species described above

were also observed in spectra of chelator-treated as-iso-

lated preparations, but an additional high-spin heme signal

was also observed, with g = (6.4, 5.4) and E/D = 0.021.

This signal was observed either alone (Fig. 4, spectrum B)

or overlapped with the g = 6.0 feature (Fig. 4, spec-

trum 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, spec-

trum D). A microwave power study at 10 K indicates that

the sharp giso = 2.00 signal is easily saturated at less than

1 lW. The other signals in the spectrum begin to saturate

at powers greater than 80 lW.

A fourth signal with one principal g value near 2.08 can

also be observed in many preparations; however, the other

associated g values are poorly resolved at 10 K. Spectral

overlap became less problematic at 130 K, as this signal

remains slow-relaxing, while the gave = 1.94 and 1.90 sig-

nals are broadened at that temperature; spectra collected at

that temperature suggest that the other features associated

with the g = 2.08 resonance are near g = 1.99, affording

Fig. 5 High-field X-band EPR spectra of intact mitochondria. A Non-

chelator-treated, as-isolated, B a more recent preparation of non-

chelator-treated, as-isolated (200 lW), C EDTA treated, NO-exposed

(9.458 GHz, 200 lW, gain 5.02 · 104), D EGTA-treated, O2-

exposed, E EGTA-treated, oxidized with 1 mM ferricyanide. Other

conditions were as for Fig. 4 except that the average microwave

frequency was 9.43 GHz. The intensities of B–E have been multiplied

by 5, 5, 2, and 5, respectively. Microwave power in A, D, and E was

200 lW

Table 2 Electron paramagnetic resonance (EPR) signals observed from whole mitochondria from Saccharomyces cerevisiae

Signal Spin state parameters g values

(g1, g2, g3)

Concentration

range (lM)

Tentative assignment

High-spin Fe3+ heme 1 S = 5/2, E/D = 0.041 6.9, 5.0 0–3 Cytochrome c peroxidase (Ccp1p)

High-spin Fe3+ heme 2 S = 5/2, E/D = 0.021 6.4, 5.4 0–2 Cytochrome c oxidase, heme a3:Cub

High-spin Fe3+ 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

system

2.085, 1.989, 1.985 1–20 Unassigned; possibly spin-interacting

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

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

123

gave = 2.02 for the signal. This was confirmed by spectral

simulation and decomposition. The 10 K and 200 lW

spectrum of an EGTA-treated sample was decomposed

(Fig. 6, spectrum A, solid line) by simulating the gave =

1.90 (Fig. 6, spectrum B), 1.94 (Fig. 6, spectrum C), 2.00

(Fig. 6, spectrum D) and 2.02 (Fig. 6, spectrum E) signals,

using g values listed in Table 2. The intensity of each

simulation was adjusted to produce a sum of the four sim-

ulations (Fig. 6, spectrum A, dashed line) that best matched

the experimental spectrum. Experimental spectra from

other preparations gave similar deconvolutions.

EPR of intact mitochondria treated with various

redox agents

Earlier mitochondrial preparations that had been exposed

to air for 1–2 days exhibited low-field regions essentially

devoid of heme-containing signals. More recent prepara-

tions, exposed to O2 for 6 h, exhibited the high-spin heme

signal at g = (6.4, 5.4) at high concentration (Fig. 4,

spectrum B). In these samples, the g = 2 region generally

consisted of intense signals with gave = 2.01 and giso =

2.00, and were largely devoid of gave = 1.94 and 1.90

signals (e.g., Fig. 5, spectrum D). A similar set of signals

were observed in a sample oxidized with ferricyanide. In

this case, the low-field region displayed a mixture of the

g = 6.0 and g = (6.4, 5.3) high-spin heme signals, while

the high-field region revealed an intense gave = 2.01 signal

(spin concentration approximately 5 lM) (Fig. 5, spec-

trum E) along with a sizable giso = 2.00 signal (1 lM) and

low-intensity gave = 1.94 and 1.90 signals.

The gave = 2.01 signal was not observed in spectra of

samples treated with dithionite or in spectra of most as-

isolated preparations, indicating that the species exhibiting

this signal is EPR-silent when reduced. The low-field

region of spectra from samples treated with dithionite was

largely devoid of signals, as expected from the thermody-

namic ability of dithionite to reduce Fe3+ hemes. The

gave = 1.94 and 1.90 signals were present, as expected, but

with concentrations similar to that observed in as-isolated

samples. The reduction ability of dithionite declines as pH

is lowered [102], and we anticipated that the intensity of

these signals might increase significantly at pH 8.5 relative

to the intensity at pH 7.4. This expectation was not ful-

filled. However, an unresolved absorption-like feature at

g * 6.4 was observed in spectra of a sample reduced with

dithionite at pH 8.5 (Fig. 4, spectrum E) but not at pH 7.4

(Fig. 4, spectrum D). This may be associated with S = 3/2

[Fe4S4]+ clusters [103].

Another preparation was exposed to 1 atm NO. This

afforded a signal at g\ = 2.07 and a g|| = 2.01 resonance

that exhibited a 14N hyperfine splitting of a = 14 G (Fig. 5,

spectrum C). This signal is characteristic of a pentacoor-

dinate heme–nitrosyl complex [101]. The spin concentra-

tion associated with this signal (20 lM) was quite high,

and it may reflect the overall concentration of pentacoor-

dinate Fe2+ heme species present, as such species are

known to bind to NO to yield similar signals.

Mossbauer spectra of mitochondria

For readers not familiar with details of Mossbauer spec-

troscopy we have given a short tutorial-type section in the

supplementary material. For the present study we have

collected Mossbauer spectra from numerous samples of

intact mitochondria. A spectrum from an as-isolated sam-

ple not exposed to metal chelators, shown in Fig. 7, spec-

trum A, exhibits three distinct spectral features (similar

spectra were observed for preparations treated with metal

chelators, EGTA and EDTA). Approximately 15–20% of

the iron belongs to a doublet with quadrupole splitting

DEQ & 3.3 mm/s and isomer shift d & 1.3 mm/s; this

doublet is outlined in the experimental spectrum. The

quoted values are typical of high-spin mononuclear Fe2+

ions in penta- or hexacoordinate nitrogen/oxygen envi-

ronments: FeII(H2O)6 complexes, the iron sites in reduced

dioxygenases and iron superoxide dismutase, and of fully

reduced binuclear iron-oxo centers at low applied field.

High-spin Fe2+ hemes have distinctly smaller d values

(approximately 0.83–0.93 mm/s); however, such species

would be difficult to resolve if they were to account for less

than 5% of the Fe in the present samples.

A second doublet in Fig. 7, spectrum A (outlined as the

dashed line in Fig. 7, spectrum B), accounting for 55–65%

of the iron, has DEQ & 1.15 mm/s and d & 0.46 mm/s.1

This doublet most probably represents Fe4S4 clusters in the

2+ core oxidation state. In this state [Fe4S4]2+ clusters have

a ground state with cluster spin S = 0. Low-spin Fe2+ he-

mes such as cytochromes b and c have very similar DEQ

and d values and thus their contributions would be difficult

to separate from those of [Fe4S4]2+ clusters. In principle,

the cytochromes should be oxidizable and thus detectable

by EPR; however, no such signals were identified in the

analogous samples examined by EPR. Thus, we suspect

that low-spin Fe2+ hemes do not contribute substantially to

1 For a purified Fe4S4 ferredoxin the area under the doublet can be

quantified to within 1–2%. Here, the uncertainties are considerably

larger, primarily because more than one cluster contributes. The

primary contributors to the doublet may be aconitase and dihydroxy-

acid dehydratase. Because species with slightly different but

unresolved parameters contribute, lineshapes are heterogeneously

broadened Lorentzians. We used both the Lorentzian and the Voight

lineshape options of WMOSS. As Voight shapes are narrower at the

base, this option yields, upon visual inspection, a lower estimate for

the concentration.

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

123

this doublet. We comment further on the DEQ = 1.15 mm/s

component when we discuss the spectrum of Fig. 7,

spectrum C.

A third component present in Fig. 7, spectrum A

exhibits broad absorption extending over a velocity range

of roughly 10 mm/s; this feature reflects unresolved mag-

netic hyperfine structure of (mostly) high-spin Fe3+ ions as

well as other unidentified low-spin magnetic species.

Finally, as much as 12% of the total iron may belong to

S = 1/2 [Fe4S4]+ clusters (discussed below).

In principle, Mossbauer spectroscopy can be used, with

some effort and proper calibration, to determine the abso-

lute 57Fe concentration of a sample. We have done this for

many years, mainly for keeping track of 57Fe enrichment in

proteins, which we have calibrated with ferredoxins and

dioxygenases. Thus, with our equipment, a 5-mm-thick

frozen aqueous solution sample containing 1 mM 57Fe

exhibiting a quadrupole doublet of 0.30 mm/s full width at

half maximum yields 5% resonance absorption. Using this

empirical rule (comparing the total absorption area to that

under the ‘‘standard’’ doublet), the sample of Fig. 7, spec-

trum A has a 57Fe concentration of approximately 0.5 mM.

A similarly prepared sample (Fig. 7, spectrum B), but

treated with the chelator EGTA, has an 57Fe concentration

of approximately 0.3 mM (both calculations taking into

effect the solvent void volume). Although unsupplemented

with Fe, the media in which the yeast were grown certainly

contained some natural-abundance Fe; thus, these Moss-

bauer spectroscopy-based estimates may be somewhat

1

0

-5 0 5Velocity (mm/s)

0.5

0.0

1

0

Abs

orpt

ion

(%)

1

0

A

B

C

D

8 T

Fig. 7 Low-temperature (4.2 K) Mossbauer 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

comprising predominantly [Fe4S4]2+ clusters. The solid line repre-

sents the sum of the Fe2+ and [Fe4S4]2+ components. C Same as for Bbut 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 lineabove the data is a simulation for a high-spin Fe3 component, and the

solid line drawn through the data is the sum of the Fe3+ and [Fe4S4]2+

species. The Fe2+ component was not simulated because this would

require use of many unknown parameters. D The 45-mT spectrum of

mitochondria treated with O2/antimycin. The solid line outlines the

contributions of the DEQ = 1.15 mm/s doublet (52%) and the Fe2+

component (12%)

Fig. 6 Simulations of the EPR spectrum of Fig. 5, spectrum B for as-

isolated mitochondria without chelator. A Data (solid line) and sum

(dashed line) of simulations (B–E). The g values of the simulations

are stated for species with gave and concentrations of B 1.90, 44 lM;

C 1.94, 23 lM; D 2.00, 2 lM; E 2.02, 17 lM. Experimental

conditions are the same as for Fig. 5, spectrum B

J Biol Inorg Chem (2007) 12:1029–1053 1039

123

lower than those obtained by chemical analysis since

Mossbauer spectroscopy only detects 57Fe in our samples.

The EGTA-treated sample (Fig. 7, spectrum B) contains

essentially the same spectral components as the sample that

was not treated with chelators (Fig. 7, spectrum A); how-

ever, the proportions were somewhat different, with 40–

50% in the DEQ = 1.15 mm/s doublet, approximately 20%

high-spin Fe2+, 15% high-spin Fe3+ ions and some as yet

unidentified iron. These percentages for the samples dis-

cussed in this study are summarized in Table 3. Figure 7,

spectrum C was recorded at 4.2 K in the presence of an

external magnetic field of 8.0 T applied parallel to the

c beam. The central feature, outlined separately by the

dashed line, belongs to the feature assigned to [Fe4S4]2+

clusters. This simulation was generated with the assump-

tion that the DEQ = 1.15 mm/s doublet represents iron

residing in a diamagnetic (S = 0) environment, in good

agreement with the data. The two absorption bands at +8

and �8 mm/s Doppler velocity belong to high-spin Fe3+

species with N/O octahedral coordination; a spectral sim-

ulation (solid line) is shown above the data. This compo-

nent, representing approximately 15% of the 57Fe, is

probably a collection of various mononuclear Fe3+ species

with octahedral N/O ligation; such species typically have

zero-field splitting parameters |D| < 2/cm and isotropic

magnetic hyperfine coupling constants A0 & �(27–29)

MHz.2

Figure 8 shows 4.2 K Mossbauer spectra of mitochon-

dria treated with 10 mM dithionite at pH 8.5; the spectra

were recorded in parallel applied fields of 50 mT (Fig. 8,

spectrum A) and 8.0 T (Fig. 8, spectrum B). For this

sample approximately 45% of the 57Fe is found to be

associated with the DEQ = 1.15 mm/s doublet. Compared

with the Fig. 7, spectrum A, there is increased absorption

(from various paramagnetic species) around �1.8 and

+2.2 mm/s Doppler velocity (arrows). These features most

probably belong to S = 1/2 [Fe4S4]+ clusters. The solid

lines overlapping the data in Fig. 8 are simulations typical

of S = 0 [Fe4S4]2+ clusters (drawn as the dashed lines to

represent 45% of total Fe) and from S = 1/2 [Fe4S4]+

(40%) clusters; the latter are, somewhat arbitrarily, repre-

sented by two cluster forms using the parameters of

reduced aconitase (20%) and a (generic) set of parameters

similar to those of the [Fe4S4]+ cluster E. coli sulfite

reductase (20%). These two spectral components which

have been used to represent the [Fe4S4]+ state could also be

drawn into Fig. 7, spectrum A, each representing 10% of

the 57Fe in that spectrum. Interestingly, with the present

decomposition, approximately 85% of the 57Fe would

belong to Fe4S4 clusters in the dithionite-reduced sample.

This estimate is probably a bit high, as some of the

absorption attributed to [Fe4S4]+ clusters may result from

[Fe2S2]+ clusters (but not more than 12%; see next para-

graph). Additional absorption attributed to [Fe4S4]2+ clus-

ters may also arise from low-spin Fe2+ cytochromes. In a

low external field, oxidized Fe3S4 clusters would be present

as an S = 1/2 absorption extending beyond the central

[Fe4S4]2+ doublet. While we see no direct evidence for the

presence of such species, they could be present at con-

centrations below 5% of the total 57Fe.

We attempted to oxidize anaerobically isolated mito-

chondrial samples by exposing them to air for 1–2 days.

Such treatment had little effect on Mossbauer spectral

features, and we suspected that this redox-buffering ability

was related to the functioning of the respiratory electron

transport chain. In an attempt to block this chain and thus

prevent cytochrome oxidase from reducing O2, we treated a

sample with antimycin A, a potent inhibitor of cytochrome

bc1 [105], and then exposed it to O2. The spectrum of this

Table 3 Summary of Mossbauer and EPR results

Fe center O2/antimycin As-isolated,

no chelator

As-isolated,

EGTA

Dithionite-treated EPR

S = 0 [Fe4S4]2+ + low-spin Fe2+ 52–57% 55–65% 40–50% 45%

S = 1/2, 3/2 [Fe4S4]+ <8% <12% 40% to minor 6% (gave = 2.02)

S = 2, 1/2 [Fe3S4]0/+ <5% <5% <5% <5% 3% (gave = 2.01)

S = 0 [Fe2S2]2+ <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

2 In weak applied fields, the lowest three Kramers doublets of the

spin sextet are generally populated at 4.2 K, yielding three Mossbauer

spectra per site. Moreover, under these conditions the magnetic

splittings, like the effective g values observed by EPR, are very

sensitive to the rhombicity parameter E/D. Consequently, the high-

spin Fe3+

ions in our sample produce broad and barely discernible

features in weak fields. However, the 8.0-T spectra are fairly

insensitive to D and E/D, because the large Zeeman splitting puts

essentially all Fe3+ ions into the MS = �5/2 state, facilitating

detection and quantification.

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

123

sample (Fig. 7. spectrum D) exhibited the DEQ = 1.15

mm/s doublet with an intensity representing 52–57% of

total 57Fe in the sample. The major difference between

Fig. 7, spectra A and D is a decline from approximately

20% in Fig. 7, spectrum A to 12% in Fig. 7, spectrum D of

the high-spin Fe2+ species. An 8.0-T spectrum (not shown)

revealed that 5% of the total iron of the sample giving

Fig. 7, spectrum D is high-spin Fe3+.

Because the central region of the spectrum of the

O2/antimycin A treated sample is comparatively clean, we

studied this sample in an expanded velocity scale. This

allowed us to search for the presence of [Fe2S2]2+ and

[Fe2S2]+ clusters. Because of spectral crowding and the low

signal amplitudes, we do not have unequivocal evidence

for the presence of this cluster type, but we can give some

upper limits. We are quite confident that less than 5% of

the total iron belongs to (diamagnetic) [Fe2S2]2+ clusters.

Up to 12% of the iron may belong to conventional and

Rieske-type [Fe2S2]+ clusters, indicating that no more than

approximately 17% of the total iron in these samples arises

from Fe2S2 clusters. Finally, only 8% of the total absorp-

tion may belong to [Fe4S4]+ clusters. These estimates could

be improved by studying matched samples with both EPR

and Mossbauer spectroscopy, and we plan to do this in the

future.

Discussion

The objectives of this study were to estimate (1) the con-

centration of Fe in mitochondria; (2) the distribution of that

Fe into various structural groups (heme, Fe/S clusters, etc.)

and (3) the degree to which the redox state of these groups

could be altered by treating intact mitochondria with redox

agents. Our approach was to obtain EPR and Mossbauer

spectra of whole intact mitochondria. We consider the

study to be exploratory and more qualitative than is nor-

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

precision typical of a purified metalloprotein. Second, it is

impossible to deconvolute spectra into individual and

assignable protein components. We explored whether

obtaining even approximate and qualitative information

relevant to these objectives would provide new insights

into how Fe is metabolized within these organelles.

Metal and protein concentrations

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

the fraction of the volume due to the mitochondria them-

selves. Our calculations assumed that samples were devoid

of impurities and that none of the radioactive sucrose used

in the experiment moved into the mitochondria. Sucrose is

commonly used to match the osmotic pressure of mito-

chondrial buffers to that within the organelles, a property

that implies the inability of sucrose to penetrate mito-

chondrial membranes. Electron micrographs and fluores-

cence images of our preparations did not reveal significant

contamination. The values obtained (approximately 800 lM

Fe by chemical analysis and approximately 500 lM 57Fe

by Mossbauer spectroscopy, and approximately 70 mg/mL

protein) have relative uncertainties of about ±25%, as

assessed from repeated measurements.

As far as we are aware, all previously reported metal ion

contents of mitochondria have been in terms of ratios of Fe

concentrations to protein concentrations, typically given in

units of nanomoles of Fe per milligram of mitochondrial

protein (which in our case is approximately 10 nmol Fe/mg

protein). Given the complexity of the organelle, it may not

be possible to fully rationalize the ratio we obtained with

other reported ratios. Cobine et al. [57] measured 2.3 nmol

0.4

0.2

0.0

-10 -5 0 5 10

Velocity (mm/s)

0.4

0.2

0.0

Abs

orpt

ion

(%)

A

B

a

a

b

b

8 T

Fig. 8 Mossbauer spectra at 4.2 K of dithionite-treated mitochondria

at pH 8.5. Spectra were recorded in 50 mT (A) and 8.0 T (B) parallel

applied fields. The dashed line outlines the contribution (45%) of

species contained in the DEQ = 1.15 mm/s doublet. The solid linesdrawn 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 (b20%). The solid lines drawn through the data are the sum of the three

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

J Biol Inorg Chem (2007) 12:1029–1053 1041

123

Fe/mg for mitochondria biosynthesized under respiratory

conditions when the growth medium was not supplemented

with Fe, and 13 nmol Fe/mg when the medium was sup-

plemented with Fe (our medium was not supplemented). It

does not appear that metal chelators were added during

isolation. The nearly sixfold observed difference suggests

that the Fe-to-protein concentration ratio depends sensi-

tively on the Fe concentration of the growth medium, and

perhaps on other factors. Because these are ratios, differ-

ences may be due to changes in either Fe concentration

and/or protein concentration. Other reported values range

between 2.5 and 5 nmol Fe/mg mitochondrial protein

[106, 107]. Tangaras et al. [108] reported 4.3 nmol

Fe/mg, divided roughly into 20% heme, 50% Fe/S clusters

and 30% ‘‘non-heme non-FeS’’. Wallace et al. [6] reported

1.2 nmol Fe/mg, while Kispal et al. [109] reported

2 nmol/mg‘‘free’’ Fe (i.e., non-heme non-Fe/S).

With use of in vivo fluorescence, the absolute concen-

tration of chelatable Fe within mitochondria from rat

hepatocyte and endothelial cells has been estimated to be

between 4.8 and approximately 12 lM [110, 111]. This

should represent Fe on the inside of mitochondria but not

tightly associated with proteins. Mitochondria from human

fibroblasts and lymphoblasts contain 1–2 lM of such

chelatable Fe [112]. If similar concentrations of such Fe

were present in our samples (approximately 8 lM), this

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

mononuclear high-spin Fe. We also caution that this refers

to chelatable Fe within the organelle, and is not related to

the chelatable Fe which is responsible for the difference in

Fe concentrations observed with/without added chelators.

Anaerobic isolation

We isolated yeast mitochondria under anaerobic conditions

to afford better control of the redox status of these organ-

elles. Anaerobic isolation was also a precautionary mea-

sure, because a number of mitochondrial proteins are

inactivated by exposure to excess O2 or by the effects of

oxidative stress. For example, iron is imported into the

matrix and delivered to the scaffold proteins in the reduced

Fe2+ state [113]. Similarly, copper ions appear to be

imported in the reduced cuprous state. In vitro Fe/S bio-

synthesis requires anaerobic conditions [114], biotin syn-

thase is inactivated by O2 [18] and maximal ferrochelatase

activity is observed under anaerobic conditions [115].

Under oxidizing conditions, the labile iron in the Fe4S4

cluster of aconitase dissociates into an Fe3S4 cluster,

thereby inactivating the enzyme [116]. With the exception

of the IM ferrochelatase, these O2-sensitive enzymes are

located in the matrix (however, ferrochelatase receives

Fe2+ ions from the matrix). Henze and Martin [117] and

Muhlenhoff and Lill [118] suggest that the matrix is the

most anaerobic compartment in O2-respiring cells. Given

the predominant role of O2 in reactions occurring within

mitochondria, this might seem counterintuitive. However,

the O2-consuming reactions occur at the IM which

encapsulates the matrix, and these reactions may occur fast

enough to effectively remove any O2 that diffuses into the

matrix.

In the oxidized inactivated state, the [Fe3S4]+ cluster of

aconitase affords an EPR signal with gave = 2.01 [5, 119],

similar to that observed here. The Fe3S4 cluster of succ-

cinate dehydrogenase exhibits a similar signal in the oxi-

dized state [2]. This signal was absent in all but one of the

as-isolated samples, which may have been slightly oxidized

relative to other as-isolated samples. The more intense Fe3+

heme signals exhibited by this particular batch relative to

the other as-isolated samples are congruent with this pos-

sibility. The absence of the gave = 2.01 signal in spectra of

our anaerobically prepared samples (and in spectra of

dithionite-reduced mitochondria) as well as the presence of

this signal under oxidizing conditions indicate that this

signal probably arises from an oxidized [Fe3S4]+ cluster,

either from inactivated aconitase, homoaconitase or the

[Fe3S4]+ cluster of succinate dehydrogenase.

Adventitious Fe and Mn

The hyperfine-split signal observed in the g = 2 region of

various as-isolated samples is typical of adventitious or

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-

ing proteins in yeast mitochondria, Sod2p and Mtm1p,

are present at concentrations below our EPR detection

limit. Sod2p is a matrix-localized manganese superoxide

dismutase while Mtm1p is an IM manganese chaperone

[58]. Based on S = 5/2 spin Hamiltonian simulations using

the known parameters for Sod2p, namely, D = 0.348/cm

and E/D = 0.026 [119, 121], our calculations indicate that

we could detect a minimum concentration of 10 lM Mn-

Sod2p. Thus, we suspect that Mn-Sod2p is present at a

concentration less than this.

The g = 4.3 signal probably arises from Fe3+ ions that

are adventitiously bound to mitochondrial proteins or

membrane phospholipids. Our studies show that this type

of iron can be removed by EDTA and EGTA, suggesting

that it resides in a region that can be accessed by these

chelators, such as the outer face of the OM or perhaps the

IMS. In more recent preparations, the amount of this

adventitious Fe3+ appears to be minimal, even in samples

1042 J Biol Inorg Chem (2007) 12:1029–1053

123

prepared in the absence of chelators. The presence of

aqueous Fe3+ ions in the OM or the IMS suggests that this

region has an electrochemical potential sufficiently high to

support this state. Previous reports have used the g = 4.3

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

this signal may also arise from Fe3+ peripherally associated

with these organelles.

Species containing Fe/S clusters

The gave = 1.94 signal was a reproducible spectral feature

of all as-isolated and dithionite-reduced samples. We ten-

tatively assign this signal to the [Fe2S2]+ cluster of succi-

nate dehydrogenase. This cluster, when reduced, exhibits

an EPR signal with g values nearly identical to those

observed here [123]. A similar signal in other reported

mitochondrial preparations has been observed and assigned

similarly [77]. However, the assignment should be cau-

tiously accepted because gave = 1.94 signals are charac-

teristic of both [Fe4S4]+ and [Fe2S2]+ containing proteins

and we cannot exclude the possibility that the observed

signal arises from one or more [Fe4S4]+ clusters rather than,

or in addition to, the [Fe2S2]+ cluster in Sdh2p.

Further support for assigning the gave = 1.94 signal to the

[Fe2S2]+ cluster of succinate dehydrogenase comes from the

saturation properties of the gave = 1.94 signal (P1/2 = 57 mW

at 10 K), which are similar to those reported for the succinate

dehydrogenase [Fe2S2]+ cluster under conditions where the

Fe4S4 cluster in the same enzyme is reduced to the para-

magnetic 1+ state [3]. When the Fe4S4 cluster is oxidized to

the diamagnetic 2+ state, the saturation behavior of the

[Fe2S2]+ cluster differs substantially (P1/2 = 0.63 mW). The

observed saturation behavior suggests that the Fe4S4 cluster

is reduced to the 1+ core oxidation state in our samples, and

spectral features due to this species should be a component of

the Mossbauer spectra shown in Fig. 7. Our inability to

observe the broad EPR features reported for the [Fe4S4]+

cluster of Sdh2 is not surprising as these low-intensity fea-

tures are easily missed [3]. Since E�0 for the [Fe4S4]2+/+

couple is�270 mV [124, 125], this implies that the potential

of the solution for which this cluster is in redox-equilibrium

is at or below this value.

The gave = 1.90 signal is similar to that exhibited by the

isolated Rieske [Fe2S2]+ protein [4], and we assign it as

such. It should contribute to the magnetic components of

Fig. 7. E�0 for the 2+/1+ redox couple of this cluster is

+280 mV [126]. This protein is part of the cytochrome bc1

complex, which is located in the IM, but the Rieske protein

itself is tethered to the rest of the complex and extends into

the IMS. This cluster transfers electrons to the IMS protein

cytochrome c, suggesting that it is in redox-equilibrium

with the IMS. Assuming this, the presence of this signal

suggests that the potential of the IMS is less than +280 mV

in our samples.

Species containing hemes

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

(E/D * 0) most likely arise from cytochrome c oxidase.

Signals with identical g values have been reported to arise

from the heme a3:Cub active site in an intermediate redox

state in which Cub is reduced to the 1+ state, while heme a3

is high-spin Fe3+ [70, 127, 128]. These signals are observed

for samples that were oxidized by O2 or ferricyanide (and

in spectra from the single ‘‘as-isolated’’ batch that exhib-

ited the gave = 2.01 signal and was slightly more oxidized

than the others). The occurrence of this signal probably

requires mildly oxidizing conditions, in that exposure to O2

in our protocol was followed by a relatively slow anaerobic

packing procedure during which time some re-reduction

could have occurred. The absence of these high-spin Fe3+

signals in the dithionite-treated samples is consistent with

the ability of dithionite to reduce Fe3+ heme a3. This

behavior is also consistent with a reduction potential for the

Fe3+/Fe2+ heme a3 site of approximately +350 mV [129].

In EPR studies of isolated cytochrome c oxidase, the

combined quantified intensity of these signals corre-

sponded to 23–50% of the cytochrome c oxidase concen-

tration [70, 127, 128]. Since the maximum combined spin

concentration observed here for these signals was approx-

imately 3 lM, these percentages suggest a minimum

cytochrome c oxidase concentration in mitochondria of 6–

13 lM. The combined heme a plus heme a3 concentration

in mitochondria has been estimated at 0.15–0.3 lmol/g

mitochondrial protein [130], which suggests a cytochrome

c oxidase concentration (assuming 70 mg/mL protein

concentration) of 5–10 lM, close to what we observe by

spin quantification. The intense signal that developed upon

treating mitochondria with nitric oxide undoubtedly arose

from pentacoordinate heme–nitrosyl groups [104] and it

indicates a minimum heme concentration in our samples of

20 lM. A significant contribution to this signal is likely

from NO binding to the Fe2+ heme a3 of cytochrome c

oxidase. Considered collectively, we suspect that the con-

centration of cytochrome c oxidase in mitochondria from

respiring yeast is between 6 and 20 lM.

The third high-spin Fe3+ heme signal (g = 6.8, 5.0;

E/D = 0.042) probably originates from heme b in cyto-

chrome c peroxidase (Ccp1p), as similar g values have

been reported [131, 132]. The particular degree of rhombic

J Biol Inorg Chem (2007) 12:1029–1053 1043

123

distortion in this heme depends on pH and subtle structural

changes. The redox potential for the Fe3+/Fe2+ couple of

this IMS protein is �182 mV at pH 7 [133]. If this signal

arises from Ccp1p, the potential of the IMS region in our

samples would appear to be greater than this value.

Alternatively, this Fe3+ heme signal may originate from

matrix-localized catalase A (Cta1p) or flavohemoprotein

(Yhb1p) as the Fe3+ states of these proteins exhibit similar

g values [134, 135]. A similar signal from mitochondria

from Spodeptera littoralis was assigned to catalase [77].

However, we expect that the potential of the matrix would

be sufficiently low to reduce these centers fully.

EPR signals from low-spin Fe3+ hemes are typically

found at gz = 3.7–2.4 and gy = 2.5–2.1 [136], but no rec-

ognizable signals were found in this region. Signals from

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

number of such groups in mitochondria (e.g., cytochromes

b, b2, c, c1, heme a), many of which should be in redox-

equilibrium with the IMS. Previous studies reported con-

centrations of 0.2–0.4 lmol/g protein for cytochrome c and

0.07–0.25 lmol/g protein for cytochrome c1 [130], both of

which correspond to easily detectable concentrations in our

samples with 70 mg/mL protein. With an estimate of

0.8 lM for the concentration of flavocytochrome b2

(Table 4), this protein would also be detectable. Since

E�0 = +290 mV for cytochrome c [137], +230 mV for

cytochrome c1 [138], �3 mV for flavocytochrome b2 [139]

and +255 mV for heme a [140], it seems likely that the

potential of the IMS in our samples was below approxi-

mately 0 mV, rendering these centers ferrous and

EPR-silent.

Organic radical species

The isotropic giso = 2.00 signal has g values and saturation

properties typical of organic radicals, and we assign this

signal to the population of such radicals in our samples.

Possible sources include the semiquinone states of flavins

and ubiquinone, and conceivably reactive oxygen species.

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

concentrations of the giso = 2.00 signal were so low. This

circumstance may have arisen because our mitochondria

were isolated anaerobically such that any radical species

generated during cell growth could have decayed during

the lengthy isolation period. The increased intensity of the

giso = 2.00 signal for samples prepared under oxidizing

conditions supports this possibility and highlights the

importance of preparing these organelles anaerobically.

An unassigned mitochondrial EPR signal

Our samples exhibited an EPR signal with a distinct res-

onance at g = 2.08 and having gave = 2.02. Positive fea-

tures near g = 2.08 are usually associated with Fe4S4

clusters, however our signal does not show the corre-

sponding higher-field features typical of such clusters;

rather, the partners for this species appear to be in the 2.00

region. Broader signals at or near g = 2.08 have been

assigned to a spin-coupled cluster involving the reduced S2

cluster of complex II (succinate CoQ oxidoreductase) [3,

142] and we have considered assigning it as such. We have

also considered assigning it to ETF dehydrogenase [25–

27]. Given the uncertainties, we leave this signal unas-

signed pending further study.

Absence of Cu2+-based signals

We did not observe signals characteristic of Cu2+ ions,

even though, by chemical analysis, our samples contained

copper at detectably high concentrations. The lack of such

signals suggests that the vast majority of Cu in our samples

is in the diamagnetic Cu+ state. The most well-known Cu

centers in mitochondria are the CuA and CuB sites in

cytochrome c oxidase. Oxidized CuA exhibits an EPR

signal with g = 2.17 [143], but no such signal was obvi-

ously present. E�0 for CuA is +240 mV [144]. This center

should be in redox-equilibrium with the IMS, as it func-

tions by accepting electrons from cytochrome c. Since

there is a detectable concentration of cytochrome c oxidase

in our samples, this implies that the absence of a signal

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

in the diamagnetic Cu+ state [145, 146]. The majority of Cu

in yeast mitochondria appears unassociated with proteins

and located in the matrix in a Cu+ form [57]. Our results

are consistent with this, both in terms of the concentration

of Cu observed and the absence of Cu2+-based EPR signals.

However, we remain puzzled why no Cu2+ signals were

observed under oxidizing conditions.

Electrochemical potentials of mitochondrial

compartments

It would be difficult to interpret our EPR results by

assuming that all redox centers in our mitochondrial sam-

ples sensed the same electrochemical potential. Given the

likelihood that the [Fe2S2]+ cluster of succinate dehydro-

genase was observed, with saturation properties indicating

that the Fe4S4 cluster of this enzyme was also reduced, the

1044 J Biol Inorg Chem (2007) 12:1029–1053

123

Ta

ble

4Ir

on

-co

nta

inin

gp

rote

ins

inm

ito

cho

nd

ria

fro

mS

.ce

revi

sia

e

Pro

tein

Gen

eC

op

ies

per

cell

Co

nce

ntr

atio

n

(lM

)

Lo

cati

on

Pro

sth

etic

gro

up

E�0

(mV

,N

HE

)E

lect

ron

ican

dm

agn

etic

pro

per

ties

Cy

toch

rom

ec

iso

form

Icy

c17

,73

01

.2IM

S[4

6]

Hem

ec

+2

90

[13

7]

S=

1/2

Fe3

+(g

=3

.06

,2

.26

,1

.25

)[3

2]

and

S=

0F

e2+

Cy

toch

rom

ec

iso

form

IIcy

c71

,31

00

.2IM

S[4

6]

Hem

ec

+2

86

[15

6]

S=

1/2

Fe3

+(g

=3

.2,

2.0

5,

1.3

9)

[32]

and

S=

0

Fe2

+

Cy

toch

rom

ec

per

ox

idas

ecc

p1

6,7

30

1.1

IMS

[15

7]

Hem

eb

�1

82

[13

3]

S=

5/2

Fe3

+(g

=6

.60

,5

.23

,5

-CN

;an

d

g=

6.1

3,

5.8

1,

6-C

N)

[15

8]

and

S=

2F

e2+

Fla

vo

cyto

chro

me

b2

cyb

24

,59

00

.8IM

S[1

57]

Hem

eb

2�

3[1

39]

S=

1/2

Fe3

+(g

=2

.99

,2

.22

,1

.47

)[1

59]

and

S=

0F

e2+

Cy

toch

rom

eb

c 1ri

p1

––

IM(f

acin

gIM

S)

[28,

15

5]

Fe 2

S2

(Rie

ske)

+2

85

[12

6,

16

0]

S=

0[F

e 2S

2]2

+an

dS

=1

/2[F

e 2S

2]+

(g=

2.0

2,

1.9

0,

1.8

0)

[4]

Cy

toch

rom

eb

c 1cy

t13

9,9

00

6.6

IM(r

edo

xw

ith

rip

1)

[28

,1

55]

Hem

ec 1

+2

30

(est

)[1

38]

S=

1/2

Fe3

+(g

=3

.33

or

3.3

5)

[16

1,

16

2]

and

low

-sp

inF

e2+

Cy

toch

rom

ec

ox

idas

eco

x1–

–IM

[16

3]

Hem

ea

+3

20

[31]

S=

1/2

Fe3

+(g

=3

.03

,2

.21

,1

.45

)[1

27,

16

4]

and

low

-sp

inF

e2+

Cy

toch

rom

ec

ox

idas

eco

x1–

–IM

[16

3]

Hem

ea

3:C

ub

+3

50

[12

9]

Fu

lly

ox

idiz

ed:

EP

R-s

ilen

tF

e3+

spin

-co

up

led

to

Cu

2+

wit

hJ*

1/c

m.

Inte

rmed

iate

:S

=5

/2

Fe3

+(g

=6

.4,

5.3

)[1

27]

mix

edw

ith

(g=

6.0

)

wh

enC

u+.

Fu

lly

red

uce

d:

hig

h-s

pin

Fe2

+:C

u+

[16

4,

16

5]

Su

ccin

ate

deh

yd

rog

enas

esd

h3

:sd

h4

23

8:7

,92

00

.04

:1.3

IM[1

]H

eme

b+

60

[16

6]

(bu

tth

isis

for

no

n-S

cen

zym

ew

hic

h

has

no

vel

Cy

s)

S=

1/2

Fe3

+(g

=3

.63

)[1

67]

and

S=

0F

e2+

Cy

toch

rom

eb

c 1co

b1

––

IM[2

8,

15

5]

Hem

eb

H�

45

(�3

5to

+2

5)

[15

5]

S=

1/2

Fe3

+(g

=3

.45

)[1

62]

and

S=

0F

e2+

Cy

toch

rom

eb

c 1co

b1

––

IM[2

8,

15

5]

Hem

eb

L�

15

0(�

95

)[1

55]

S=

1/2

Fe3

+(g

=3

.78

)[1

62]

and

S=

0F

e2+

Fer

roch

elat

ase

hem

15

22

,70

03

.8IM

(fac

ing

M)

[42]

Mo

no

nu

clea

rF

e–

S=

2F

e2+

(d=

1.3

6m

m/s

;D

EQ

=3

.04

mm

/s)

[11

5]

Su

ccin

ate

deh

yd

rog

enas

eS

dh

29

,54

01

.6IM

(fac

ing

M)

[1]

Fe 2

S2

0[3

]S

=0

[Fe 2

S2]2

+an

dS

=1

/2[F

e 2S

2]+

(g=

2.0

26

,

1.9

35

,1

.91

2)

[3,

16

6]

Su

ccin

ate

deh

yd

rog

enas

eS

dh

29

,54

01

.6IM

(fac

ing

M)

[1]

Fe 3

S4

+6

0[3

]S

=1

/2[F

e 3S

4]+

(g=

2.0

1)

and

S=

2[F

e 3S

4]0

[16

6]

Su

ccin

ate

deh

yd

rog

enas

eS

dh

29

,54

01

.6IM

(fac

ing

M)

[1]

Fe 4

S4

�2

60

[3]

S=

0[F

e 4S

4]2

+an

dS

=1

/2[F

e 4S

4]+

(g=

2.0

64

,

1.9

92

,1

.84

7an

dm

agn

etic

inte

ract

ion

s

affo

rdin

gfe

atu

res

at2

.27

and

1.6

3)

[3]

Hem

em

on

oo

xy

gen

ase

cox1

5–

–IM

[35

]H

eme

a[3

4,

16

8]

+2

42

[16

8]

S=

1/2

Fe3

+(g

=3

.5)

and

S=

0F

e2+,

[16

8]

Hem

em

on

oo

xy

gen

ase

cox1

5–

–IM

[35

]H

eme

b[3

4,

16

8]

+8

5[1

68]

S=

1/2

Fe3

+(g

=3

.7)

and

S=

0F

e2+,

[16

8]

Car

bo

xy

late

mo

no

xy

gen

ase

Co

q7

––

IM[1

69]

Fe–

O–

Fe

[48

,1

70]

+4

8an

d�

13

5[1

71]

(Pu

tati

ve)

S=

0[F

e2+

Fe2

+],

S=

1/2

[Fe3

+F

e2+]

(g=

1.9

5,

1.8

6,

1.7

7)

and

S=

4[F

e2+

Fe2

+]

[17

2]

ET

Fd

ehy

dro

gen

ase

YO

R3

56

W(p

uta

tiv

e)

3,3

20

0.6

IMF

e 4S

4[2

6]

+4

7[2

7]

S=

0[F

e 4S

4]2

+an

dS

=1

/2[F

e 4S

4]+

(g=

2.0

86

,

1.9

39

,1

.88

6)

[25]

J Biol Inorg Chem (2007) 12:1029–1053 1045

123

Ta

ble

4co

nti

nu

ed

Pro

tein

Gen

eC

op

ies

per

cell

Co

nce

ntr

atio

n

(lM

)

Lo

cati

on

Pro

sth

etic

gro

up

E�0

(mV

,N

HE

)E

lect

ron

ican

dm

agn

etic

pro

per

ties

Aco

nit

ase

Aco

19

6,7

00

16

M[1

73]

Fe 4

S4

and

Fe 3

S4

�4

50

,�

26

8,

+1

00

[17

4]

S=

0[F

e 4S

4]2

+an

dS

=1

/2[F

e 4S

4]+

(g=

2.0

6,

1.9

3,

1.8

6)

[5]

S=

1/2

[Fe 3

S4]+

(g=

2.0

24

,

2.0

16

,2

.00

4)

and

S=

2[F

e 3S

4]0

S=

1/2

[Fe 4

S4]3

+an

dS

=0

[Fe 4

S4]2

+

Ho

mo

aco

nit

ase

Lys

47

,35

01

.2M

[6]

Fe 4

S4

and

Fe 3

S4

(pu

tati

ve)

Sim

ilar

toac

on

itas

e[6

]S

imil

arto

aco

nit

ase

[6]

Fer

red

ox

inY

ah

11

4,8

00

2.4

M[7

]F

e 2S

2�

35

3[8

]S

=0

[Fe 2

S2]2

+an

dS

=1

/2[F

e 2S

2]+

(g=

2.0

24

,

1.9

37

,1

.93

7)

[8]

Fe/

Ssc

affo

ldp

rote

inIs

u1

10

,80

01

.8M

[22]

Fe 2

S2

(Pro

bab

lylo

w)

S=

0[F

e 2S

2]2

+[1

75]

Fe/

Ssc

affo

ldp

rote

inIs

u2

3,4

20

0.6

M[2

2]

Fe 2

S2

(Pro

bab

lylo

w)

S=

0[F

e 2S

2]2

+[1

75]

Fe/

Ssc

affo

ldp

rote

inIs

a1

12

50

.02

M[2

3]

Fe 2

S2

(Pro

bab

lylo

w)

S=

0[F

e 2S

2]2

+[1

75]

Fe/

Ssc

affo

ldp

rote

inIs

a2

1,5

60

0.3

M[1

74]

or

IMS

[23]

Fe 2

S2

(pu

tati

ve)

[17

6]

(Pro

bab

lylo

w)

S=

0[F

e 2S

2]2

+[1

75]

Fe/

Ssc

affo

ldp

rote

inN

fu1

11

,30

01

.9M

[22]

Fe 2

S2

(Pro

bab

lylo

w)

S=

0[F

e 2S

2]2

+[1

75]

Fe/

Ssc

affo

ldp

rote

inIs

u1

10

,80

01

.8M

[22]

Fe 4

S4

(Pro

bab

lylo

w)

S=

0[F

e 4S

4]2

+[1

75]

Fe/

Ssc

affo

ldp

rote

inIs

u2

3,4

20

0.6

M[2

2]

Fe 4

S4

(Pro

bab

lylo

w)

S=

0[F

e 4S

4]2

+[1

75]

Fe/

Ssc

affo

ldp

rote

inIs

a1

12

50

.02

M[2

3]

Fe 4

S4

(Pro

bab

lylo

w)

S=

0[F

e 4S

4]2

+[1

75]

Fe/

Ssc

affo

ldp

rote

inIs

a2

1,5

60

0.3

M[1

76]

or

IMS

[23]

Fe 4

S4

(pu

tati

ve)

[17

6]

(Pro

bab

lylo

w)

S=

0[F

e 4S

4]2

+[1

75]

Fe/

Ssc

affo

ldp

rote

inN

fu1

11

,30

01

.9M

[22]

Fe 4

S4

(Pro

bab

lylo

w)

S=

0[F

e 4S

4]2

+[1

75]

Bio

tin

syn

thas

eB

io2

50

40

.08

M[1

77]

Fe 4

S4

�4

40

[17

8]

S=

0[F

e 4S

4]2

+an

dS

=1

/2[F

e 4S

4]+

(g=

2.0

42

,

1.9

37

,1

.93

7)

[18]

or

(g=

2.0

35

,1

.93

7,

1.9

37

)

[14

]o

r(g

=2

.04

4,

1.9

44

,1

.91

4an

dS

=3

/2)

[17

9]

Lip

oic

acid

syn

thas

eL

ip5

1,6

30

0.3

M[1

80]

Fe 4

S4

�5

05

[18

1]

S=

0[F

e 4S

4]2

+an

dS

=1

/2[F

e 4S

4]+

(g=

2.0

39

,

1.9

37

,1

.93

7)

[18

]

Bio

tin

syn

thas

eB

io2

50

40

.08

M[1

77]

Fe 2

S2

�1

40

[17

8]

S=

0[F

e 2S

2]2

+an

dS

=1

/2[F

e 2S

2]+

(g=

2.0

1,

1.9

6,

1.8

8an

dg

=2

.00

,1

.94

,1

.85

)[1

4,

18

2]

Lip

oic

acid

syn

thas

eL

ip5

1,6

30

0.3

M[1

80]

Fe 2

S2

�4

30

[18

1]

S=

0[F

e 2S

2]2

+an

dS

=1

/2[F

e 2S

2]+

Dih

yd

rox

yac

idd

ehy

dra

tase

Ilv3

17

1,0

00

28

M(p

uta

tiv

e)[1

9]

Fe 4

S4

(pu

tati

ve)

(Dit

hio

nit

e-re

du

cib

le)

[20

]

S=

0[F

e 4S

4]2

+an

dS

=3

/2[F

e 4S

4]+

(g=

5.2

,

4.7

)[1

9,

20]

Fra

tax

inh

om

olo

gY

fh1

1,5

60

0.3

M[1

83]

2m

on

on

ucl

ear

Fe’

s

[18

4]

(Pro

bab

lyh

igh

)S

=5

/2F

e3+

and

Fe2

+[1

84,

18

5]

Cat

alas

eA

Cta

16

23

0.1

M[3

7]

Hem

eb

�2

26

(est

[13

4])

S=

5/2

Fe3

+(g

=6

.48

,5

.10

)[1

86]

Fla

vo

hem

og

lob

inY

hb

11

3,0

00

2.2

M[1

35]

(an

d

cyto

sol)

Hem

eb

�2

30

to�

32

0(e

st[1

87])

S=

5/2

Fe3

+(g

=5

.75

,6

.47

,5

.22

)[1

88]

NH

En

orm

alh

yd

rog

enel

ectr

od

e,IM

Sin

term

emb

ran

esp

ace,

IMin

ner

mem

bra

ne,

Mm

atri

x

1046 J Biol Inorg Chem (2007) 12:1029–1053

123

potential of the solution with which this cluster is in redox-

equilibrium should be less than approximately �270 mV.

These clusters of succinate dehydrogenase should be in

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

most likely from the IMS cytochrome c peroxidase, and the

lack of a signal from flavocytochrome b2 suggests that it is

in the reduced state. This implies an IMS solution potential

in the range from �200 to 0 mV in our samples. If we

assume a potential difference between the IMS and the

matrix of 180 mV [147], an IMS potential range of �100

to �200 mV would predict a matrix potential range of

�300 to �400 mV, which are both compatible with our

observations.

Mossbauer spectroscopy

To date, only one Mossbauer spectrum of wild-type mito-

chondria has been published and it was devoid of any

signals [39]. In contrast, a sample of the mutant Dyfh1

displayed a quadrupole doublet with DEQ = 0.67 mm/s and

d = 0.52 mm/s which was assigned to amorphous nano-

particles of iron(III) phosphate; other Fe-containing com-

ponents were not observed. We have recorded Mossbauer

spectra of more than 25 preparations of intact mitochon-

dria, over the course of 2 years and involving various

group members preparing these samples. Also, the Moss-

bauer 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 Moss-

bauer 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 Mossbauer spec-

troscopy are complementary. EPR detects with high sen-

sitivity species with half-integral spin (Kramers systems),

while Mossbauer spectroscopy, in this first study, is mainly

useful in the exploration of components with integer or

zero electronic spin, i.e., components either not accessible

(diamagnetic complexes) or only difficult to access by

EPR. We consider our present Mossbauer spectroscopy

results to be preliminary, but we believe that the proven

power of the technique can be exploited in the future, once

the system is dissected by metabolic and/or genetic

manipulations, e.g., overexpression or deletion of particu-

lar mitochondrial proteins. Our present studies suggest that

one should conduct the Mossbauer spectroscopy studies at

4.2 K and in weak as well as in strong applied fields. A

strong-field capability is essential as it allows one to

identify S = 0 species such as [Fe4S4]2+ and [Fe2S2]2+

clusters. Spectra recorded in strong applied fields distin-

guish also between monomeric high-spin Fe3+ and nano-

particles containing high-spin Fe3+.

Approximate iron distribution in mitochondria

from respiring yeast

Our Mossbauer spectroscopy and EPR results are insuffi-

cient to establish precisely how Fe ions in mitochondria are

distributed, but they are sufficient to allow us to draw some

approximate and preliminary conclusions, which are sum-

marized by the pie chart shown in Fig. 9. The majority of Fe

in mitochondria from respiring yeast is present as Fe4S4

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-

ters are in the 2+ state, but a substantial fraction can be

reduced to the 1+ state by incubation of mitochondria with

dithionite at pH 8.5. Thus, in Fig. 9 we distinguish dithio-

nite-reducible [Fe4S4]2+/+ clusters from irreducible

[Fe4S4]2+ clusters. The next most abundant class of Fe-

containing species in mitochondria, representing approxi-

mately 20% of the Fe in Fig. 9 (but with an acceptable range

Fig. 9 Comparison of observed and calculated percentile Fe distri-

bution. Percentages used in the pie chart for the observed distribution

were as follows: 37% ([Fe4S4]2+ + low-spin Fe2+ hemes), 25%

[Fe4S4]2+/+, 22% high-spin (Fe3+ + Fe2+) non-heme mononuclear, 9%

[Fe2S2]2+/+, 4% high-spin hemes (Fe3+ + Fe2+) and 3% [Fe3S4]+/0

clusters. These values are based on our results, collectively consid-

ered, but should be viewed as a hypothesis, with substantial latitude in

our estimates for each category. Calculated percentages were 78%

[Fe4S4]2+/+, 5% low-spin hemes (Fe2+ only), 5% high-spin Fe3+/2+

non-heme mononuclear, 8% [Fe2S2]2+/+, 2% high-spin hemes (Fe3+/2+)

and 2% [Fe3S4]+/0. These values are taken from Table S1, assuming

8 lM nonproteinatious high-spin non-heme Fe2+ ions. The concen-

tration of Fe associated with each Fe-containing mitochondrial

protein was calculated and percentages were obtained by dividing

each individual Fe concentration by the sum of all such values and

multiplying by 100. HS low spin, LS low spin

J Biol Inorg Chem (2007) 12:1029–1053 1047

123

of 15–35%), is high-spin non-heme Fe. In the as-isolated

state, most of these ions are high-spin Fe2+, with smaller

proportions of high-spin Fe3+. We have not observed low-

spin Fe3+ ions in any of our samples. Our EPR results

suggest that approximately 9% (but with a range of 5–11%,

as limited by our Mossbauer spectroscopy analysis) of the

Fe is present as [Fe2S2]+ clusters, whereas no such clusters

in the 2+ state were observed. The DEQ = 1.15 mm/s

doublet, attributed essentially to [Fe4S4]2+ clusters, may

contain contributions from low-spin ferrous hemes. The

remaining few percent of mitochondrial Fe is present as

high-spin heme and perhaps Fe3S4 centers.

The spectral simulations of Fig. 8 suggest that the main

part of the magnetic features represent S = 1/2 [Fe4S4]+

clusters. While we are reasonably certain that the magnetic

features reflect paramagnetic Fe/S clusters, we do not wish

to suggest that the entire magnetic component belongs to

the S = 1/2 forms (the gave = 1.94 species) of [Fe4S4]+

clusters; we suspect that S = 3/2 [Fe4S4]+ as well as

[Fe2S2]+ clusters, albeit to a lesser extent, also contribute.

By EPR, we see signals assigned to these latter species

(S = 3/2 [Fe4S4]+ and S = 1/2 [Fe2S2]+ clusters). By

studying the Mossbauer and EPR spectra of aliquots of the

same dithionite-treated sample, one should be able to

assess the cluster type and concentration of the reduced Fe/

S species better.

Regarding high-spin ferrous species, many hexa-and

pentacoordinated complexes with N/O ligation contribute

doublets with DEQ = 3.0–3.5 mm/s and d = 1.3–1.4 mm/s.

With a few exceptions the high-field spectra of these

complexes are broad and difficult to analyze.

Included in the [Fe4S4]2+ cluster portion are clusters that

convert into Fe3S4 clusters upon oxidation (e.g., aconitase);

this fraction could represent as much as approximately

25 lM Fe (3% of the total). Treatment with dithionite at

pH 8.5 causes approximately half of the [Fe4S4]2+ portion

to become reduced to the [Fe4S4]+ state.

Mossbauer spectra of as-isolated mitochondria indicate

that approximately 15% of the Fe is paramagnetic and in

half-integer spin states. According to our EPR results, this

would include approximately 3 lM due to the high-spin

heme signal from cytochrome c peroxidase/catalase,

approximately 3 lM due to the high-spin heme signals

from cytochrome c oxidase, approximately 20 lM due to

g = 4.3 high-spin Fe3+, approximately 40 lM due to the

[Fe2S2]+ cluster of the Rieske protein and approximately

20 lM due to the [Fe2S2]+ cluster from succinate dehy-

drogenase. The [Fe4S4]+ cluster of succinate dehydroge-

nase might also contribute to the paramagnetic component.

Summing these EPR contributions affords approximately

90 lM Fe, translating into approximately 11% of the total

Fe, in qualitative agreement with what is observed by

Mossbauer spectroscopy.

Comparison with known Fe-containing proteins

in mitochondria

We have organized known mitochondrial proteins start-

ing with the results of three proteomic studies [148–150],

the information provided by the Saccharomyces Ge-

nomics Database and the reconstructed metabolic net-

work of Forster et al. [151]. The integration of this

information led to the identification of approximately 600

candidate mitochondrial proteins, a number comparable

to the approximately 800 proteins estimated to constitute

the complete yeast mitochondrial proteome [150].

Primary research literature describing properties of each

of these proteins was accessed using the Web of Science

(http://www.isi10.isiknowledge.com) and information

specifically regarding Fe content and suborganellar

localization was sought.

The result of this analysis afforded the proteins and pro-

tein complexes included in Table 4. It is difficult to establish

that this or any such list is complete, and we suspect that

there are Fe-containing mitochondrially localized proteins

that are not included. Some such proteins might be uniden-

tified currently, or the presence of Fe in them might be

uncertain. Also not included in this list are proteins that are

known to interact with Fe (e.g., transporters) but for which

no Fe-bound state has been characterized.

The concentrations of many of these proteins within the

mitochondria have been estimated. Ghaemmaghami et al.

[153] created a comprehensive fusion library of S cerevisiae

cells in which each member had a different open reading

frame tagged with the same epitope. Natural expression

levels of the corresponding fusion proteins were quantified

to afford copy numbers per cell (Table 4). The volume of an

S. cerevisiae cell is approximately 1 · 10�13 L, and mito-

chondria occupy approximately 10% of this [154]. Thus,

one copy of a mitochondrial component per cell reflects a

concentration of 170 pM in the organelle. Such information

can be useful in interpreting Mossbauer spectra.

It is interesting to compare this list with the results

observed in this study. We calculated the overall Fe con-

centration in mitochondria implied by the proteins and

concentrations given in this table, by summing the products

of the concentration of each known Fe-containing mito-

chondrial protein (Ci) and the number of Fe’s per protein

(mi); i.e., [Fe]overall ¼Pn

i¼1 miCi (if the number of copies

per cell was not reported, a concentration of 1 lM was

assumed). The calculated overall concentration of Fe in

mitochondria was 265 lM (Table S1), corresponding to

only one third of our experimentally determined value. If

correct, this suggests that two thirds of the detected Fe in

yeast mitochondria is not accounted for by Table 4,

assuming the protein concentrations in that table. On the

other hand, systematically low estimates of protein

1048 J Biol Inorg Chem (2007) 12:1029–1053

123

concentration in the literature might also be responsible for

this discrepancy. Tempered by this caveat, some interesting

trends are apparent, namely:

• A large proportion of Fe in mitochondria appears to be

associated with the Fe4S4 cluster from a single protein,

namely, dihydroxyacid dehydratase.

• The OM is devoid of Fe-containing proteins.

• The matrix contains few heme-containing proteins

(only catalase and flavohemoglobin).

• The matrix is dominated by Fe/S centers, especially

[Fe4S4]2+ clusters.

• Only one mitochondrial protein with an Fe–O–Fe

center is known (Coq7p).

In order to compare our experimental EPR and Mossbauer

spectroscopy results with predictions made from these

calculations, two additional pieces of information for each

site are required, namely, the region of the mitochondria

with which the site is in redox-equilibrium and the solution

potential of that region under the conditions when our

samples were frozen. For some redox centers (e.g., cyto-

chrome c), there is no doubt as to the region with which they

are in redox-equilibrium (e.g., the IMS), but such informa-

tion is not certain for all entries in Table 4. Nor is the

solution potential for each region of the mitochondria

(under the specific conditions for which our samples were

prepared) known. For proteins located in either aqueous

region (IMS or matrix), the region with which they were

assumed to be in redox-equilibration was the region where

the proteins were located. For proteins located in the IM,

there were a number of possibilities. Some sites extend into

either the IMS or the matrix, and if such sites are known to

accept/donate electrons with donors/acceptors in that

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

complexes might be along a known electron pathway which

leads to either aqueous region, and such sites were deemed

to be in redox-equilibrium with that aqueous region. In a

few cases, redox-dependent protein conformation changes

occur such that sites contained within those proteins might

be in redox-equilibrium with more than one region,

depending on the protein conformation at the time our

samples were frozen, and so no definitive assignment could

be made. Finally, the active site for cytochrome c oxidase

(heme a3:Cub) might be unique in being in redox-equilib-

rium with the O2/H2O couple (E� * +800 mV). For our

samples prepared under anaerobic conditions, the appro-

priate non-standard-state reduction potential to use would

be much less than +800 mV. Our set of tentative assign-

ments is given in Table S1.

Next, we estimated the solution potential of the IMS and

matrix to be EIMS & �0.1 and Ematrix & �0.3 V,

respectively (see discussion above). EIM probably lies

somewhere between these two values and may be con-

trolled by the E� value for the CoQox/CoQred couple,

namely, +60 mV in the IM [155]. What must separate the

redox potential of one region from another and maintain

regions in redox isolation are the redox-dependent con-

formational changes known for the IM-bound respiratory

complexes.

Then, we grouped all species of Table 4 that would give

rise to equivalent Mossbauer spectral features, and calcu-

lated the concentration of Fe associated with each group.

This procedure resulted in the nine groups listed in

Table S1. The calculated percentages of mitochondrial Fe

in various forms are also shown in Fig. 9. Comparison with

what we have observed in this study indicates overall

qualitative agreement. The greatest apparent discrepancy is

the greater percentage of high-spin non-heme Fe observed

in our samples relative to that predicted by the calculations.

The qualitative similarities in the calculated versus

observed distribution of Fe in mitochondria indicates a

general agreement between our results and the calculated

contents of these organelles. Calculations predict that there

should be fewer high-spin Fe2+ ions and more ([Fe4S4]2+

clusters + low-spin Fe2+ hemes) than we observe. Some of

the observed high-spin Fe2+ ions may be adventitiously

bound, despite our attempt to remove such ions by chela-

tion. Alternatively, some Fe-containing mitochondrial

proteins have not been included in the calculations, or a

portion of these ions might represent a transient and che-

latable ferrous ion pool, as has been reported previously

[111]. In this latter case, the concentration of ions esti-

mated for this pool (2–12 lM Fe) would be substantially

less than we observe (approximately 180 lM).

Conclusions

The major contributions of this study are:

1. Protein and metal concentrations. We determined the

absolute concentrations of protein and Fe in ‘‘neat’’

(solvent-free) yeast mitochondria, synthesized by

respiring cells and isolated in the presence of metal

chelators to be approximately 70 mg/mL and 800 ±

200 lM, respectively.

2. EPR signals from proteins containing Fe/S cluster and

heme prosthetic groups. Signals were observed that

have been tentatively assigned to succinate dehydro-

genase, the Rieske Fe/S protein, aconitase, cytochrome

c oxidase and cytochrome c peroxidase. An intense

signal with gave = 2.02 was observed. Although

unassigned, this signal probably originates from an

Fe/S cluster.

J Biol Inorg Chem (2007) 12:1029–1053 1049

123

3. Species not observed. No signals from Cu2+ ions, Mn2+

superoxide dismutase or low-spin Fe3+ hemes were

observed. These species may be present at undetecta-

bly low concentrations or in EPR-silent states. The

collective concentration of organic-based radical spe-

cies was unexpectedly low.

4. Electrochemical compartmentalization. In the as-iso-

lated state of our samples, the ranges of the potentials

for the IMS and the matrix are �0.2 < EIMS < �0.1 V

and �0.4 < Ematrix < �0.3 V (vs NHE), respectively.

5. Dominance of Fe4S4 clusters. The majority of Fe (50–

85%) in mitochondria isolated from respiring cells is

present in this form.

6. High-spin ferrous ions. Approximately 20% of the Fe

in mitochondria is present as high-spin non-heme Fe2+

ions with five to six O/N ligands coordinating. A

portion of this is probably adventitiously bound to the

mitochondria, while the remainder is associated with

mitochondrial proteins and/or conceivably a ‘‘pool’’ of

such ions, perhaps in the mitochondrial matrix.

7. Fe2S2 clusters. A modest proportion of the Fe

(approximately 5–10%) in mitochondria is present as

these types of clusters, including those from succinate

dehydrogenase and the Rieske Fe/S protein.

8. Hemes. A similarly modest proportion of Fe in

mitochondria is present as heme prosthetic groups.

9. Distribution of Fe in mitochondrial regions. The OM

is largely devoid of Fe-containing proteins, while the

matrix is dominated by Fe4S4 clusters.

Note added in proof: It now appears less likely that Isa1p and Isa2p

contain Fe/S clusters similar to those in other Fe/S scaffold proteins.

Acknowledgements We thank the following people: Art Johnson

and Holly Cargill (Department of Biochemistry and Biophysics,

Texas A&M University) for instructions on isolating mitochondria;

Rola Barhoumi (Image Analysis Laboratory, Texas A&M University)

and Anne Ellis (Microscopy and Imaging Center, Texas A&M Uni-

versity) for collecting microscopic images; Jinny Johnson (Protein

Chemistry Laboratory, Texas A&M University) for performing amino

acid analyses; David P. Giedroc (Department of Biochemistry and

Biophysics, Texas A&M University) for access to his atomic

absorption spectrophotometer; William James (Department of

Chemistry, Texas A&M University) for training on and assistance

with the inductively coupled plasma mass spectrometer; Shelly

Henderson Possi for help in isolating some batches and in measuring

O2 consumption; Tanner Freeman for preparing one of the EPR

samples; and Roland Lill for helpful discussion.This study was sup-

ported by the Robert A. Welch Foundation (A1170) and The National

Institutes of Health [GM077387 (M.P.H.), EB001475 (E.M.) and The

Chemistry Biology Interface training program (B.N.H. and J.G)].

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