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CONTRIBUTORS TO VOLUME 349 xi

KELLEY KININGHAM(30), Graduate School JOE M. McCoRD (33), Webb-Waring Insti-


of Toxicology, University of Kentucky, tute, University of Colorado Health Sci-
Lexington, Kentucky 40536 ence Center, Denver, Colorado 80262
LARS-OLIVER KLOTZ (10), Institute for KEVIN R. MESSNER (36), Department
Physiological Chemistry I, Heinrich- of Microbiology, University of Illinois
Heine-Universitiit Diisseldorf, D-40225 at Urbana-Champaign, Urbana, Illinois
Diisseldorf, Germany 61801
WILLEM H. KOPPENOL (12), Laboratory of YASUHIDEMIYAMOTO(14), Department of
Inorganic Chemistry, Eidgeniissische Biochemistry, Osaka University Medical
Technische Hochschule, CH-8093 Zurich, School, Osaka 565-0871, Japan
Switzerland
ROBIN J. MOCKETT (21, 28), Department
J. SIMON KROLL (16), Molecular Infectious of Molecular Pharmacology and Toxicol-
Diseases Group, Department of Paedi- ogy, University of Southern California,
atrics, Imperial College of Science, Tech- Los Angeles, California 90033
nology and Medicine, St. Mary's Hos-
pital Campus, London W2 1PG, United ISABEL MOURA (24), Department of Chem-
Kingdom istry and Center for Chemistry and
Biotechnology, FCT Universidade Nova
LINDA K. KWONG (34), Department of de Lisboa, 2825-114 Monte de Caparica,
Molecular Pharmacology and Toxicol- Lisbon, Portugal
ogy, University of Southern California,
Los Angeles, California 90033 Josl~ J. G. MOURA (24), Department of
Chemistry and Center for Chemistry and
PAUL R. LANGFORD (16), Molecular In- Biotechnology, FCT Universidade Nova
fectious Diseases Group, Department of de Lisboa, 2825-114 Monte de Caparica,
Paediatrics, Imperial College of Science, Lisbon, Portugal
Technology and Medicine, St. Mary's
Hospital Campus, London W2 1PG, ALl NAINI (19), Department of Neurol-
United Kingdom ogy, Columbia University, New York,
New York 10032
JIN-WON LEE (9), Laboratory of Biophysics,
School of Biological Sciences, and Insti- HARRY S. NICK (6), Department of Neuro-
tute of Microbiology, Seoul National Uni- science, University of Florida College of
versity, Seou1151-742, Korea Medicine, Gainesville, Florida 32610

MURIELLE LOMBARD (13), Laboratory of MANABU NISHIKAWA(35), Department of


Chemistry and Biochemistry of Bio- Biochemistry and Molecular Pathology,
logical Redox Centers, DBMS-CEA/ Osaka City University Medical School,
CNRS/Universitg Joseph Fourier, 38054 Osaka 545-8585, Japan
Grenoble Cedex 9, France
VINCENT NIVI~RE (13), Laboratory of
KENSAKU MAEDA (35), Department of Chemistry and Biochemistry of Bio-
Biochemistry and Molecular Pathology, logical Redox Centers, DBMS-CEA/
Osaka City University Medical School, CNRS/Universit~ Joseph Fourier, 38054
Osaka 545-8585, Japan Grenoble Cedex 9, France
STEEAN L. MARKLUND (7), Department of PETER O'NEILL (4), Radiation and Genome
Medical Biosciences, Clinical Chemistry, Stability Unit, Medical Research Coun-
Ume& University Hospital, SE-901 85 cil Harwell, Didcot, Oxon OXllORD,
Ume&, Sweden United Kingdom
xii CONTRIBUTORS TO VOLUME 349

TOMOMI OOKAWARA(14), Department of DARET K. ST. CLAIR (30), Graduate Cen-


Biochemistry, Osaka University Medical ter for Toxicology, University of Ken-
School, Osaka 565-0871, Japan tucky, Lexington, Kentucky 40536
WILLIAM C. ORR (21), Department of Bio- AMRAM SAMUNI (23), Department of
logical Sciences, Southern Methodist Molecular Biology, School of Medicine,
University, Dallas, Texas 75275 Hebrew University of Jerusalem,
AH-MEE PARK (35), Department of Bio- Jerusalem 91120, Israel
chemistry and Molecular Pathology, ASSUNTA SANSONE (16), The European
Osaka City University Medical School, Bioinformatics Institute, EMBL Outsta-
Osaka 545-8585, Japan tion, Wellcome Trust Genome Campus,
SUREERUT PORNTADAVITY (30), Gradu- Cambridge CBIO 1SD, United Kingdom
ate Center for Toxicology, University of
Kentucky, Lexington, Kentucky 40536 EISUKESATO(35), Department of Biochem-
istry and Molecular Pathology, Osaka
SERGE PRZEDBORSKI (19), Departments City University Medical School, Osaka
of Neurology and Pathology, Columbia 545-8585, Japan
University, New York, New York 10032
HELMUT SIES (10), Institute of Physio-
CELIA QUIJANO (3), Department of Bio- logical Chemistry, Heinrich-Heine-Uni-
chemistry, Universidad de la Repdblica, versit?it Diisseldo~ D-40225 Diisseldo~
11800 Montevideo, Uruguay Germany
RAFAEL RADI (3), Department of Biochem-
DAVID N. SILVERMAN (6), Department
istry, Universidad de la Reptlblica, 11800
Montevideo, Uruguay of Pharmacology, University of Florida
College of Medicine, Gainesville, Florida
INES RAINERI (20), Department of Pe- 32610
diatrics, Genetics Division, University
of California, San Francisco, California BARBARA H. SOHAL (28), Department of
94143 Molecular Pharmacology and Toxicol-
ogy, University of Southern California,
HYUNE MO RHO (29), School of Biologi- Los Angeles, California 90033
cal Sciences, Seoul National University,
Seoul 151-1742, Korea RAJINDAR S. SOHAL (21, 28, 34), De-
partment of Molecular Pharmacology
JUNG-HYE ROE (9), Laboratory of Mo-
and Toxicology, University of Southern
lecular Microbiology, School of Biologi-
California, Los Angeles, California
cal Sciences, and Institute of Micro- 90033
biology, Seoul National University, Seoul
151-742, Korea RHONDAE SOUZA(31), University of Texas
NORMA ROMERO (19), Department of Neu- Southwestern Medical Center and Dallas
rology, Columbia University, New York, Veterans Administration Medical Center,
New York 10032 Dallas, Texas 75216
GIUSEPPE ROTILIO(5), Department of Biol- CHANDRA SRINIVASAN(18), Department of
ogy, University of Rome "Tor Vergata," Chemistry and Biochemistry, University
00133 Roma, Italy of California, Los Angeles, California
90095
FRANK RUSNAK (24), Section of Hematol-
ogy Research, Department of Biochem- MARIA ELENA STROPPOLO (4), National
istry and Molecular Biology, Mayo Clinic Institute for the Physics of Matter (INFM)
and Foundation, Rochester, Minnesota and Department of Biology, University of
55905 Rome "Tor Vergata," 00133 Rome, Italy
CONTRIBUTORS TO VOLUME 349 xiii

LOR! A. STURTZ (17), Division of Toxicol- LAURA VALDEZ (27), Laboratory of Free
ogical Sciences, Environmental Health Radical Biology, School of Pharmacy and
Sciences, Bloomberg School of Pub- Biology, University of Buenos Aires, I 113
lic Health, Johns Hopkins University, Buenos Aires, Argentina
Baltimore, Maryland 21205 PIERA VALENTI (16), Institute of Micro-
DAVID SULZER (19), Department of Neu- biology, H Universitgl di Napoli, 80138
rology, Columbia University, New York, Naples, Italy
New York 10032 JANY VASSY (32), Laboratory of Image
BARBARA SUTTER (12), Laboratory of ln- Analysis of Cellular Pathology, H6pital
organic Chemistry, EidgenOssische Tech- Saint Louis, 75010 Paris, France
nische Hochschule, CH-8093 Zurich, JAMES W. WHITTAKER (8), Department
Switzerland of Biochemistry and Molecular Biology,
Oregon Graduate Institute School of Sci-
KEIICHIRO SUZUKI (14), Department of
ence and Engineering, Oregon Health
Biochemistry, Osaka University Medical
and Science University, Beaverton,
School, Osaka 565-0871, Japan
Oregon 97006
NAOYUKI TANIGUCHI(14), Department of ANH N. WOODMANSEE (1), Department
Biochemistry, Osaka University Medical of Microbiology, University of Illinois
School, Osaka 565-0871, Japan at Urbana-Champaign, Urbana, Illinois
LANCE S. TERADA(31), University of Texas 61801
Southwestern Medical Center and Dallas YONG XU (30), Graduate Center for
Veterans Administration Medical Center, Toxicology, University of Kentucky,
Dallas, Texas 75216 Lexington, Kentucky 40536
DANI~LE TOUATI (15), Jacques Monod In- HAE YONG YOO (29), School of Biologi-
stitute CNRS-Universit~s Paris 6 et Paris cal Sciences, Seoul National University,
7, 75251 Paris Cedex 05, France Seou1151-742, Korea
[1] E P R QUANTITATION OF INTRACELLULAR FREE IRON 3

[ 1 ] Quantitation of Intracellular Free Iron by Electron


Paramagnetic Resonance Spectroscopy
By A N H N . WOODMANSEE a n d JAMES A . IMLAY

Background
Cells contain "free" iron, which is distinct from the tightly bound iron found
in the iron-sulfur clusters and hemes of proteins.l-6 It has been suggested that this
pool performs several productive roles, including supplying iron for the synthesis
of iron-containing enzymes, I'2 functioning in the process of cellular iron trans-
port and storage, 7-I° and contributing to the expression or repression of iron-
responsive genes. 11-13 This iron pool has not been chemically defined. Although
iron binds avidly in vitro to numerous metabolites, including citrate, peptides, and
nucleotides, its predominant physiological ligands remain unknown. Instead, free
iron is simply conceived of as the intracellular iron that is not stably integrated
into enzymes or iron-storage proteins.
Intracellular free iron is of additional interest because of its role in both
oxidative DNA damage and lipid peroxidation. 14'15 Changes in its concentra-
tion directly affect the rate of both processes. The primary experimental evi-
dence of this assertion is that cell-permeable iron chelators block these forms
of oxidative damage in cells of all types. 16'Iv Conversely, circumstances that

l R. R. Crichton and M. Charloteanx-Waters, Eur. l. Biochem. 164, 485 (1987).


2 A. Jacobs, Blood 50, 433 (1970).
3 R. J. Rothman, A. Serroni, and J. C. Faber, Mol. Pharmacol. 42, 703 (1992).
4 p. Ponka, R. W. Grady, A. Wilczynska, and H. M. Schulman, Biochim. Biophys. Acta 802, 477
(1984).
5 D. Y. Yegorov, A. V. Kozlov, O. A. Azizova, and Y. A. Vladimirov, Free Radic. Biol. Med. 15, 565
(1993).
6 R. R. Crichton and R. J. Ward, in "Iron and Human Diseases" (R. B. Laufer, ed.), p. 23. CRC Press,
Boca Raton, Florida, 1992.
7 T. Ramasarma, Free Radic. Res. Commun. 2, 153 (1986).
8 S. P. Young, S. Roberts, and A. Bomford, Biochem J. 232, 819 (1985).
9 C. G. D. Morley, K. Rewers, and A. Bezkorovainy, in "The Biochemistry and Physiology of Iron"
(P. Saltman and J. Hegenauer, eds.), p. 171. Elsevier, Amsterdam, 1982.
l0 A. Jacobs, Ciba Found Symp. 51, 91 (1977).
I I R. D. Klausner, T. A. Rouault, and J. B. Harford, Cell 72, 19 (1993).
12 A. Bagg and J. B. Neiland, Microbiol. Rev. 51, 509 (1987).
13 T. A. Rouault and R. D. Klausner, Trends Biochem. Sci. 21, 174 (1996).
14 K. Keyer and J. A. Imlay, Proc. NatlAcad. Sci. U.S.A. 93, 13635 (1996).
15 D. M. Miller, N. H. Spear, and S. D. Aust, Arch. Biochem. Biophys. 295, 240 (1992).
J6 M. Larramendy, A. C. Mello-Filho, E. A. Leme Martins, and R. Meneghini, Mutat. Res. 178, 57
(1983).
17 j. A. Imlay, S. M. Chin, and S. Linn, Science 240, 640 (1988).

Copyright2002, ElsevierScience(USA).
All rightsreserved.
METHODS1NENZYMOLOGY,VOL.349 0076-6879/02$35.00
4 SUPEROXIDEREACTIONSAND MECHANISMS [1]

increase the amount of intracellular free iron--including mutations that accel-


erate iron transport or diminish storage--raise the rates of membrane and DNA
injury. 18 Of particular interest to investigators of oxidative stress is the fact that
free iron pools enlarge when superoxide destroys the iron-sulfur clusters of a sub-
class of dehydratases, 19 thereby promoting DNA and lipid damage as an indirect
consequence. 14,20
The involvement of free iron pools in oxidative injury is spurring the develop-
ment of methods to quantitate them. One difficulty is that free iron comprises only
about 5% of total cellular iron, so that even substantial changes in the iron pool
cannot be accurately calculated from measurement of the total iron content. For
that reason, methods must be designed to specifically detect loosely bound iron.
A second problem is that intracellular free iron concentrations cannot be inferred
from measurements made on cell lysates. The disruption of cells causes, either by
chemical oxidation or dilution, sufficient damage to metalloenzymes and storage
proteins that they release abundant iron. This artifactual iron flux dominates any
subsequent measurement of soluble iron.
Several different spectroscopic techniques have appeared that circumvent these
difficulties, albeit with different sensitivities and ease of application. The first is
the use of calcein, a cell-penetrating iron chelator whose fluorescence is quenched
when it binds iron. By determining its specific fluorescence in vivo, it is possible
to obtain information about the concentration of iron available to bind it. A com-
plication of the calcein method is that in order to preserve a workable signal-to-
noise ratio, the amount of calcein added to the sample must be limited. Because
more abundant intracellular metabolites, such as citrate, effectively compete with
calcein for iron, the data from such experiments do not directly report the amount
of total free iron. Investigators have addressed this issue by infusing cells with
standard amounts of free iron. The intracellular iron concentrations that have been
arrived at by this method--0.01 # M in plant cells 21 and 0.5/zM in human eryth-
roleukemia cells2Z--are far lower than the typically 10 -5 M values reported for
bacteria and yeast, using the electron paramagnetic resonance (EPR) technique
described below.14,23 The calcein method is most facile in comparing the relative
amounts of free iron in well-matched samples for which the concentrations of
competing intracellular chelators are unlikely to vary.

18D. Touati, M. Jacques, B. Tardat, L. Bouchard, and S. Despied, J. Bacteriol. 177, 2305 (1995).
19D. H. Flint, J. F. Tuminello, and M. H. Emptage,J. Biol. Chem. 268, 22369 (1993).
20S. I. Liochevand I. Fridovich, Free Radic. Biol. Med. 16, 29 (1994).
21 S. Epsztejn, O. Kakhlon, H. Glicktein, W. Breuer, and Z. I. Cabantchik, Anal Biochem. 248, 31
(1997).
22W. Breuer, S. Epsztejn, and Z. I. Cabantchik, J. Biol. Chem. 270, 24209 (1995).
23C. Srinivasa, A. Liba, J. A. Imlay,J. S. Valentine, and E. B. Gralla, J. Biol. Chem. 275, 29187 (2000).
[ 1] E P R QUANTITATION OF INTRACELLULAR FREE IRON 5

Free intracellular ferrous iron has also apparently been visualized in vivo by
a second noninvasive technique, M/Sssbauer spectroscopy, z4-26 This method has
not been used to monitor iron homeostasis during oxidative stress, but it may yet
prove useful, particularly because it can provide information about the ligand field
of the "free" iron.
A third method, whole-cell EPR, has been used to quantitate free iron levels in
both bacteria and yeast 14'23 and is described in detail. The technique is relatively
fast and simple, has a detection limit of about 1/zM iron, and can be adapted to
discriminate between ferrous and ferric forms. In principle, this method could be
adapted for application to mammalian cells, but the authors are unaware of any
attempts to do so.

Theory
Because they are paramagnetic, both Fe z+ and Pe 3+ present EPR signals. How-
ever, the signal for Fe 2+ is much broader and weaker than that for Fe3+; thus, free
Fe 3+ can be measured directly by EPR, whereas Fe 2+ can be measured easily
only after being oxidized to Fe 3+. Ferrous iron can be converted to the ferric
form in vivo by incubating cells with the penetrating iron chelator desferriox-
amine mesylate (DF). Because DF binds Fe 3+ more strongly than Fe z+, DF lowers
the redox potential of the latter species, thereby facilitating its oxidation by the
molecular oxygen present in the sample. The consequence is that ferrous iron is
quantitatively converted to a ferric : DF chelate. This chelate exhibits a sharp, eas-
ily detectable EPR signal at g = 4.3. Prosthetic iron in enzymes does not resonate
at this g value.
A variety of controls have validated the use of DF to identify free ironJ 4
Because DF completely blocks iron-mediated DNA damage in bacteria, it ap-
pears that the DF : Fe 3+ signal includes all the iron that participates in adventitious
oxidative chemistry in the cell. DF failed to extract iron from a variety of iron-
containing enzymes, which indicates that the signal does not represent iron that had
been integrated into proteins. Further, the exposure of bacterial cells to millimolar
amounts of H 2 0 2 yielded an EPR signal of the same intensity as when cells were
exposed to DF, indicating that the sole effect of DF is to stimulate the oxidation
of the pool of free ferrous iron. 27 H 2 0 2 therefore provides an alternative method
of oxidizing intracellular iron in those organisms that DF cannot penetrate easily.

24 T. H. Abdul, A. J. Hudson, Y. S. Chang, A. R. Timms, C. Hawkins, J. M. Williams, E M. Harrison,


J. R. Guest, and S. C. Andrews, J. Bacteriol. 181, 1415 (1999).
25 H. Abdul-Tehrani, A. J. Hudson, Y. Chang, A. R. Timms, C. Hawkins, J. M. Williams, E M. Harrison,
J. R. Guest, and S. C. Andrews, J. Bacteriol. 181, 1415 (1999).
26 B. E Matzanke, G. I. Muller, E. Bill, and A. X. Trantwein, Eur. J. Biochem. 183, 371 (1989).
27 A. L. Nguyen and J. A. Imlay, unpublished results (2001).
6 SUPEROXIDE REACTIONS AND MECHANISMS [1]

In the absence of DF or H202 treatment, the ferric iron signal in Escherichia coli
is small, indicating that the great majority of free iron is in the reduced form. The
opposite is true in y e a s t y

Preparation of Reagents
All chemicals are from Sigma (St. Louis, MO). Laboratory deionized water is
further purified with using a Labconco (Kansas City, MO) Water Pro PS system.
All reagents should be prepared fresh for each set of experiments. Fe2(SO4)3 for
iron standards is prepared as a 100 mM stock in water. It is diluted to the appropri-
ate concentrations immediately before use, because iron at lower concentrations
quickly forms ferric hydroxides and precipitates out of solution. A stock of 0.2 M
desferrioxamine mesylate is dissolved in water, and the pH is adjusted to 8.0 with
6 M stock of KOH. Cold 20 mM Tris-HC1, pH 7.4, is maintained on ice, as is the
same buffer supplemented with 10% (v/v) glycerol.

Cell G r o w t h a n d S a m p l e P r e p a r a t i o n
Cell are grown in either rich or defined medium with vigorous shaking. Low-
density cultures require large culture volumes, so that the cell sample ultimately
contains sufficient biomass, approximately 50 mg of protein. Cultures are cen-
trifuged at 10,000 rpm for 5 min at 4 °, and the pellet is resuspended in 9 ml of the
same medium. For measurement of total free iron, 1 ml of 0.2 M D F stock is added;
for measurement of only ferric iron, the DF is omitted. The resuspended cells are
then incubated with shaking at 37 ° for 10 min. The cells are then centrifuged
again, washed with ice-cold 20 mM Tris-HC1 buffer at pH 7.4, and resuspended
in 0.2-0.4 ml of the same buffer containing 10% (v/v) glycerol. The volume of the
sample is measured, and 0.2 ml of the sample is loaded into a 3-mm quartz EPR
tube, with care to avoid the introduction of air bubbles. The sample is immediately
frozen in dry ice and stored at - 7 0 ° . It is essential that all steps be performed
as rapidly as possible to ensure the most accurate measurements. The remaining
sample can be diluted and the absorbance determined, or lysed and the protein
content assayed, in order to provide a measure of cell density for comparison with
other samples.
A standard curve is generated using solutions of Fe2(SO4)3.7H20. A stock
solution of ferric sulfate dissolved in deionized water is precisely quantitated by
dilution into 20 mM Tris-HCl-1 mM DE using E420 = 2.865 mM -1 cm -1 for
the Fe 3+ : Df complex. 28 Ferric sulfate is then diluted to concentrations that are
similar to that which will be found in the cell sample tubes--approximately 10 to
50 #M---in 20 mM Tris buffer and 10% (v/v) glycerol. The standard samples are

28 D. Y. Yegorev, A. V. Kozlov, O. A. Azizova, and Y. A. Vladimirov, Free Radic. Biol. Med. 15, 565
(1993).
[1] EPR QUANTITATIONOF INTRACELLULARFREE IRON 7

loaded into EPR tubes at the same volume used for the samples, and they are
immediately frozen at - 7 0 ° . Samples and standards may be stored indefinitely
at - 7 0 ° and reused as long as they remain frozen. Total sample preparation time
(exclusive of cell growth) is approximately 40 min.

Electron Paramagnetic Resonance Parameters


The signal is measured with a Varian (Palo Alto, CA) Century E-112 X-
band spectrophotometer equipped with a Varian TEl02 cavity and a Varian tem-
perature controller. The temperature is measured with a T-type thermocouple
fixed next to the sample and an Omega Engineering (Stamford, CT) 670/680
microprocessor-based thermocouple meter. Data are analyzed with software from
Scientific Software Services (Bloomington, IL). The EPR spectrometer settings
are as follows: field center, 1570 G; receiver gain, 2500; field sweep, 400 G;
modulation amplitude, 1.25 G; T, -125°; power, 30 mW.

C a l c u l a t i o n of F r e e I r o n C o n c e n t r a t i o n
If the EPR signals in paired samples are similar in shape, then the free iron
concentration is most accurately determined by comparing the amplitude of the
first derivative peak amplitude of the sample with that of a standard that contains
a similar iron concentration. The signal shape may vary in samples that do not
contain DF, because the ligand sphere influences the spectrum. In these cases
the free iron is quantitated by double integration. This procedure provides the
amount of iron in the sample. The actual intracellular concentration is calculated
by dividing the iron amount by the intracellular volume of the sample. The latter
can be determined by establishing the relationship between intracellular volume
and the biomass of the harvested culture. For example, 1 liter of E. coli at 1.0 OD600
(containing 190 mg of protein) represents a cytoplasmic volume of 0.5 ml. Ferric
iron is directly determined from the EPR spectra of samples that have not been
treated with either DF or H202; total iron, from the treated samples; and ferrous
iron, by subtraction of the former from the latter.

P e r f o r m a n c e a n d A p p l i c a t i o n of A s s a y
Whole cell EPR allows for the measurement of non-protein-bound, chelatable
iron inside whole cells. This chelatable "free" iron is distinct from the forms of iron
found in Fe-S clusters, in heme, or in enzymes that use iron as a prosthetic metal for
catalysis. A limitation of this method is its potential inability to distinguish free
Pe 3+ from the ferric iron core of iron storage proteins, which also present a signal
at g = 4.3. However, studies with E. coli mutants that lack storage proteins showed
that in this organism the EPR iron signals arise predominantly from free rather
than stored iron.
] I I I I I I I I I
1372 '1412 1452 1492 1532 1572 1612 1652 1692 1732
NAGNETIC FIELD (GAUSS)

FIG. l. Iron EPR signals from whole cell preparations of E. coli. Two thousand data points were
collected per scan and each spectrum was the average of 50 scans per sample. The peak intensity of the
scans was normalized to cell concentration. Top scan: AB 1157, wild type, containing approximately
12/zM intracellular free iron. Bottom scan: JI 132, the isogenic superoxide dismutase-deficient strain
[K. Keyer and J. A. Imlay, Proc. Natl. Acad. Sci. U.S.A. 93, 13635 (1996)], containing 86/zM free iron.

I I I I I I I I I I
1372 1412 1452 1492 1532 1572 1612 1652 1692 :1732
NAGNETIC FIELD (GAUSS}

FIG. 2. Top scan: AN387, wild type, containing 20/zMintracellular free iron. Bottom scan: SM 1095,
an isogenic dipicolinate-accumulating strain IS. Maringanti and J. A. Imlay, J. Bacteriol. 181, 3792
(1999)], containing 320/zM free iron.
[21 ACONITASE 9

In log-phase, aerobic, wild-type E. coli that are grown in standard laboratory


media, the basal level of free iron ranges from 15 to 30 #M, depending on growth
conditions. With the methods described here intracellular iron concentrations as
low as 1 to 10/zM can be measured, albeit with significant (1 #M) variance. Some
improvement in the signal-to-noise ratio can be achieved by increasing the number
of scans per sample (to reduce the sample noise) or by increasing the concentration
of cells in the EPR tube (to increase the signal). Nevertheless, because normal
intracellular iron concentrations approach the detection limits, increases in free
iron concentration are more easily detected than are decreases.
Typical results with superoxide-stressed cells are shown in Fig. 1.14 Figure 2
demonstrates that a strain that accumulates dipicolinate, an iron-binding metabo-
lite, also contains a high amount of "free" iron. In this example the iron was
almost certainly bound to the metabolite before the addition of DE EPR analy-
sis with such strains succeeds because DF binds iron more tightly than do most
intracellular metabolites and therefore extracts the iron from them.

Acknowledgments
This work was supported by Grant GM59030 from the National Institutes of Health. We gratefully
acknowledge the contributions of Kay Keyer, Amy Gort, and Sujatha Maringanti, previous members
of this laboratory, in the development and application of these methods.

[2] Aconitase: Sensitive Target and Measure


of Superoxide
By PAUL R. GARDNER

Introduction
0 2 - is abundantly produced in aerobic organisms, and the superoxide dismu-
tases (SODs) appear ample for the task of scavenging 0 2 - . The SODs maintain
the steady-state [02"-] at a remarkably low level of ~10 -1° M inside diverse cells
and organelles.1 This [O2"-] is roughly equivalent to 1 02"- molecule in 20 bacteria
or 5000 molecules in a much larger mammalian cell. These low, steady-state in-
tracellular 02'- levels and high levels of SOD expression are essential because of
the extreme reactivity and toxicity of 02"-.2,3

1 j. A. Imlay and I. Fridovich, J. Biol. Chem. 266, 6957 (1991).


2 j, M. McCord, B. B. J. Keele, and I. Fridovich, Proc. Natl. Acad. Sci. U.S.A. 68, 1024 (1971).
3 I. Fridovich, Science 201, 875 (1978).

Copyright2002,ElsevierScience(USA).
All tightsreserved.
METHODSIN ENZYMOLOGY,VOL.349 0076-6879/02$35.00
[21 ACONITASE 9

In log-phase, aerobic, wild-type E. coli that are grown in standard laboratory


media, the basal level of free iron ranges from 15 to 30 #M, depending on growth
conditions. With the methods described here intracellular iron concentrations as
low as 1 to 10/zM can be measured, albeit with significant (1 #M) variance. Some
improvement in the signal-to-noise ratio can be achieved by increasing the number
of scans per sample (to reduce the sample noise) or by increasing the concentration
of cells in the EPR tube (to increase the signal). Nevertheless, because normal
intracellular iron concentrations approach the detection limits, increases in free
iron concentration are more easily detected than are decreases.
Typical results with superoxide-stressed cells are shown in Fig. 1.14 Figure 2
demonstrates that a strain that accumulates dipicolinate, an iron-binding metabo-
lite, also contains a high amount of "free" iron. In this example the iron was
almost certainly bound to the metabolite before the addition of DE EPR analy-
sis with such strains succeeds because DF binds iron more tightly than do most
intracellular metabolites and therefore extracts the iron from them.

Acknowledgments
This work was supported by Grant GM59030 from the National Institutes of Health. We gratefully
acknowledge the contributions of Kay Keyer, Amy Gort, and Sujatha Maringanti, previous members
of this laboratory, in the development and application of these methods.

[2] Aconitase: Sensitive Target and Measure


of Superoxide
By PAUL R. GARDNER

Introduction
0 2 - is abundantly produced in aerobic organisms, and the superoxide dismu-
tases (SODs) appear ample for the task of scavenging 0 2 - . The SODs maintain
the steady-state [02"-] at a remarkably low level of ~10 -1° M inside diverse cells
and organelles.1 This [O2"-] is roughly equivalent to 1 02"- molecule in 20 bacteria
or 5000 molecules in a much larger mammalian cell. These low, steady-state in-
tracellular 02'- levels and high levels of SOD expression are essential because of
the extreme reactivity and toxicity of 02"-.2,3

1 j. A. Imlay and I. Fridovich, J. Biol. Chem. 266, 6957 (1991).


2 j, M. McCord, B. B. J. Keele, and I. Fridovich, Proc. Natl. Acad. Sci. U.S.A. 68, 1024 (1971).
3 I. Fridovich, Science 201, 875 (1978).

Copyright2002,ElsevierScience(USA).
All tightsreserved.
METHODSIN ENZYMOLOGY,VOL.349 0076-6879/02$35.00
10 SUPEROXIDE REACTIONS AND MECHANISMS [2]

A myriad of biomolecules are oxidized or reduced by 02'- ; however, it is often


unclear whether or how these reactions cause toxic (or beneficial) actions to cells
under normal or pathological conditions. I. Fridovich has provided comprehensive
reviews of the reactions of O2'- with biomolecules,4-6 and there have been some
notable additions to the literature.
More recent attention has focused on the reactions of O2'- with members of the
growing family of [4Fe-4S]-containing (de)hydratases, including the critical citric
acid cycle enzyme aconitase, 6-8 and with the gaseous signaling molecule and toxin
nitric oxide (NO). 9 Both reactions occur rapidly, with estimated second-order rate
constants in the range of l07 and 10 l° M -l sec -1, respectively. 1°-12 Moreover, evi-
dence indicates that these targets have a critical role in O2- toxicity in various cells
under a variety of conditions. Attack of either of these targets also has the potential
to generate more lethal secondary damage to DNA, membranes, and proteins. The
reaction of O2- with NO forms the more reactive and indiscriminant oxidant and
nitrating agent peroxynitrite,9'13 and 02'- leaches highly reactive, catalytic, and
damaging iron from these labile iron-sulfur centers. 14-17The aconitases have pro-
vided a paradigm for understanding O2- toxicity and SOD function in bacteria,
yeast, plants, and mammals and have yielded a sensitive method for measuring
changes in the picomolar steady-state levels of 0 2 - inside diverse cells and within
mitochondria. This article describes the reaction of O2- with aconitase and the
principles, practice, applications, advantages, and limitations of the "aconitase
method."

P r i n c i p l e s of A c o n i t a s e M e t h o d
Members of the family of [4Fe-4S]-containing (de)hydratases are particularly
susceptible to attack and oxidative inactivation by 02"-. 6-8,18,19 The mammalian

4 I. Fridovich, Arch. Biochem. Biophys. 247, 1 (1986).


5 I. Fridovich, J. Biol. Chem. 264, 7761 (1989).
6 I. Fridovich, Annu. Rev. Biochem. 64, 97 (1995).
7 p. R. Gardner, Biosci. Rep. 17, 33 (1997).
8 D. H. Flint and R. M. Allen, Chem. Rev. 96, 2315 (1996).
9 W. A. Pryor and G. L. Squadrito, Am. J. Physiol. 268, L699 (1995).
10 A. Hausladen and I. Fridovich, J. Biol. Chem. 269~ 29405 (1994).
11 D. H. Flint, J. E Tuminello, and M. H. Emptage, J. Biol. Chem, 268, 22369 (1993).
12 R. E. Huie and S. Padmaja, Free Radic. Res. Commun. 18, 195 (1993).
13 j. S. Beckman, T. W. Beckman, J. Chen, P. A. Marshall, and B. A. Freeman, Proc. Natl. Acad. Sci.
U.S.A. 87, 1620 (1990).
14 S. 1. Liochev and I. Fridovich, Free Radic. Biol. Med. 16, 29 (1994).
15 K. Keyer and J. A. Imlay, Proc. Natl. Acad. Sci. U.S.A. 93, 13635 (1996).
16 C. Srinivasan, A. Liba, J. A. Imlay, J. S. Valentine, and E. B. Gralla, J. Biol. Chem. 275, 29187
(2O00).
17 j. V~isquez-Vivar, B. Kalyanaraman, and M. C. Kennedy, J. Biol. Chem. 275, 14064 (2000).
18 p. R. Gardner, G. Costantino, C. Szab6, and A. L. Salzman, J. Biol. Chem. 272, 25071 (1997).
19 J.-C. Drapier and J. B. J. Hibbs, Methods EnzymoL 269, 26 (1996).
[2] ACONITASE 11

2""

Cy<

C~

Cys

FIG. 1. Reaction of O2- with the aconitase [4Fe-4S] center. The avid binding and exceptional
reactivity of 02- with the [4Fe-4S]2+ center in aconitase and related (de)hydratases are attributed
to the electrophilic character of the solvent-accessibleiron atom (Fea), the extreme nucleophilicity of
02"-, the increased oxidizing potential of 02- in the iron-liganded state, and the sensitivity of these
Fea~S bonds to oxidation and dissolution.

mitochondrial citric acid cycle enzyme aconitase and the mammalian cytosolic
iron-regulatory protein and aconitase are perhaps the best studied and most un-
derstood members of this family. The aconitases contain cubane [4Fe-4S] centers
with three irons liganded to cysteine residues. The fourth iron, Fea, is exposed to the
solvent and to attack by 0 2 - (Fig. 1). Fea is essential for the catalytic dehydration
of citrate to form the intermediate cis-aconitate and the hydration of cis-aconitate
to form isocitrate (Scheme 1). The Fea atom is thought to function as a Lewis acid
in these reactions. 8 In the absence of substrates, Fea ligands water or hydroxide.
The Michaelis constants (Km values) for citrate, cis-aconitate, and isocitrate vary
with assay conditions and enzyme source, but Km values are generally less than
0.5 mM, with the Km values for cis-aconitate and isocitrate being lower than that for
citrate. Given the relatively high concentrations of aconitase substrates measured
in cells and mitochondria, aconitase would appear to be near saturation in vivo.
0 2 ' - attacks the electrophilic solvent-exposed catalytic iron atom in the
[4Fe-4S] 2+ center, causing one electron oxidation of the iron-sulfur cluster, the
release of the exposed iron atom in the ferrous oxidation state, and the loss of

H
H2C - - C O 0 - H20 HC - - COO- H20 HOC - - COO-
l J ~ II L.. I
HOC - - COO- C -- COO- HC -- COO-
l I I
H2C -- CO0- H2C --CO0- H2C -- COO-

citrate cis-aconitate isocitrate

SCHEME1

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