J Biol Inorg Chem (2005) 10: 913–923
DOI 10.1007/s00775-005-0044-y
O R I GI N A L A R T IC L E
Raylene J. Sanchez Æ Chandra Srinivasan
William H. Munroe Æ Matthew Alan Wallace
Jacob Martins Æ Tina Y. Kao Æ Kate Le
Edith Butler Gralla Æ Joan Selverstone Valentine
Exogenous manganous ion at millimolar levels rescues all known
dioxygen-sensitive phenotypes of yeast lacking CuZnSOD
Received: 2 August 2005 / Accepted: 2 October 2005 / Published online: 8 November 2005

SBIC 2005
Abstract Yeasts lacking copper-zinc superoxide dismu-
tase (sod1D) exhibit a broad range of phenotypes, many
of which can be rescued by growth in the presence of
high levels of ionic manganese. We undertook a com-
prehensive survey of the effects of manganese on wild-
type and sod1D yeasts and found that 5 mM Mn2+
rescued all known growth-related phenotypes, such as
slow growth in air, temperature sensitivity, specific
amino acid auxotrophies, no growth in high oxygen,
poor growth in nonfermentable carbon sources, and
decreased stationary-phase survival. Iron-related phe-
notypes-elevated electron paramagnetic resonance
detectable (‘‘free’’) iron, decreased aconitase activity,
and fragmenting vacuoles-as well as zinc sensitivity were
also rescued. The activity of manganese superoxide
dismutase remained constant or was reduced when the
yeasts were grown in the presence of MnCl2, indicating
that induction of this alternative superoxide dismutase is
not the explanation. In contrast to MnCl2 treatment,
addition of two manganese-containing superoxide
dismutase mimetic compounds to the growth medium
did not provide any rescue of sod1D yeast growth but
rather had an sod1D-selective inhibitory effect at
micromolar concentrations. Mechanisms by which ionic
manganese can effect this rescue, while the mimetic
compounds do not, are discussed.
R. J. Sanchez Æ W. H. Munroe Æ M. A. Wallace Æ J. Martins
T. Y. Kao Æ E. B. Gralla (&) Æ J. S. Valentine
Department of Chemistry and Biochemistry, UCLA,
607 Charles E. Young Drive, East,
Los Angeles, CA 90095-1569, USA
E-mail: egralla@chem.ucla.edu
Tel.: +1-310-8251946
Fax: +1-310-2069880
C. Srinivasan Æ K. Le
Department of Chemistry and Biochemistry,
California State University,
Fullerton, CA 92834-9480, USA
Keywords Manganese Æ Zinc Æ Copper-zinc superoxide
dismutase Æ Oxidative stress Æ Yeast
Abbreviations EPR: Electron
paramagnetic resonance Æ ICP-AES: Inductively
coupled plasma atomic emission spectrometer Æ IMS:
Intermembrane space of mitochondria Æ Leu1p:
Isopropylmalate dehydratase Æ ROS: Reactive oxygen
species Æ SDC: Synthetic complete medium with 2%
glucose Æ SD-Lys: Defined medium lacking lysine Æ
SD-Met: Defined medium lacking methionine Æ SOD:
Superoxide dismutase Æ sod1D: Yeast lacking the
CuZnSOD gene Æ sod2D: Yeast lacking the MnSOD
gene Æ SOR: Superoxide reductase Æ Tris:
Tris(hydroxymethyl)aminomethane
Introduction
Non superoxide dismutase (SOD) manganese plays a
major role in the antioxidant defense mechanisms of
several prokaryotic organisms [1]. The phenomenon was
first noted when bacterial species that lack any detect-
able SOD enzymes were found to contain high (milli-
molar) levels of dialyzable manganese; the aerobic
organism Lactobacillus plantarum, for example, lacks
any known form of SOD enzyme and contains 30 mM
manganese, which, apparently, functionally replaces
SOD enzymes. In other bacterial systems, high levels of
non-SOD manganese have been shown to work in con-
junction with antioxidant enzymes such as SOD and
catalase as a major component of their defense systems
against reactive oxygen species [1, 2]. High levels of ionic
manganese added to the growth medium also provide a
substantial degree of rescue of the dioxygen-sensitive
phenotypes of SOD knockout Escherichia coli [3].
The molecular basis for the antioxidant action of
non-SOD manganese in prokaryotes is unknown. Early
reports suggested that non-SOD ionic manganese
914
possessed SOD activity, on the basis of the xanthine/
xanthine oxidase/cytochrome c assay [4]. However, fur-
ther studies determined that ionic manganese interferes
with this indirect assay by promoting the oxidation of
reduced cytochrome c [3]. Furthermore, direct assays
using pulse radiolysis or stopped flow techniques have
established that ionic manganese at physiological pH
has little, if any, SOD activity [5-7].
Despite the fact that ionic manganese lacks signifi-
cant superoxide dismutase activity at physiological pH,
there is considerable evidence that extracts from man-
ganese-treated cells nevertheless have a high degree of
superoxide scavenging ability [1, 3, 4, 8]. Manganous ion
is well known to react with superoxide at fast rates in
vitro to form a relatively long lived transient complex
[MnO2]+ [5, 7, 9, 10]. If this transient is rapidly reduced
in vivo by an unknown cellular reductant, non-SOD
manganese may be acting as a superoxide reductase
(SOR), functioning in a manner analogous to the non-
heme iron-containing FeSORs [11].
The possibility that non-SOD manganese functions as
an antioxidant in vivo has been less explored in eukary-
otes than in prokaryotes. Most of the evidence that exists
in support of such a function comes from studies of
Saccharomyces cerevisiae. The budding yeast S. cerevi-
siae, like most eukaryotes, contains two SODs: CuZn-
SOD (SOD1) in the cytosol, the intermembrane space of
mitochondria (IMS), and other locations, and MnSOD
(SOD2) in the mitochondrial matrix. Yeast cells lacking
CuZnSOD (sod1D) have severe aerobic growth defects,
die quickly in the stationary phase, and exhibit lysine,
leucine, and methionine auxotrophies under normal
culture conditions [12, 13]. They also exhibit altered iron
and zinc metabolism, when grown aerobically, including
elevated electron paramagnetic resonance (EPR) detect-
able iron levels at g=4.3 and zinc sensitivity [14-16]. The
most common genetic suppressors of CuZnSOD
knockout (sod1D) yeast, i.e., pmr1-1 and bsd2-1, lead to
dramatic increases in intracellular manganese levels [17,
18]. Additionally, the inability of sod1D yeast to survive
on medium lacking lysine or methionine as well as its
poor growth in 100% dioxygen have previously been
shown to be rescued by the addition of 5 mM MnCl2 to
the growth medium [8, 17].
Other metal ions, in particular copper and iron, have
also been reported to rescue certain phenotypes of sod1D
yeast when added to the growth medium, but the rescue
in each case was incomplete. Copper, working through a
mechanism that requires the presence of metallothionein,
restored the ability of sod1D yeast to grow on the non-
fermentable carbon source, lactate, but did not restore
lysine prototrophy or the ability to grow in an atmo-
sphere of 100% dioxygen [19]. Similarly, iron supple-
mentation restored growth on glycerol, but not
methionine prototrophy [16]. In addition to simple metal
ions, several classes of manganese coordination com-
plexes have been developed as functional SOD mimics
and tested for biological efficacy in different model sys-
tems. The two of particular interest to us in this study are
(1) derivatives of Mn(III) salen complexes Euk-8, Euk-
134, and Euk-189 developed by Eukarion [20] and (2)
derivatives of Mn(III) cyclam complexes M40403 and
M40404 developed by Metaphore Pharmaceuticals [21].
The Mn(III) salen (Euk) complexes have been reported
to increase wild-type Caenorhabditis elegans lifespan [22,
23] and were beneficial in other models of oxidative
stress, such as ischemia-reperfusion injury in a rat stroke
model [24], reactive oxygen species (ROS)-induced
apoptosis [25], and improved survival of sod2 null mice
[26]. The Mn(III) cyclam derivative M40403 has been
found to be efficacious in reducing ischemia-reperfusion
injury [21, 27-29], beneficial in inflammation models in
rats [30, 31], and protective of cochlear hair cells [32, 33].
The present study was undertaken to determine the
nature and degree of the manganese rescue of sod1D
yeast phenotypes and to compare the effect of ionic
manganese with that of some manganese-containing
SOD mimics that had earlier been shown to have sig-
nificant degrees of biological activity in other experi-
mental systems. On the basis of earlier studies of L.
plantarum and other bacteria entirely lacking in SOD
enzymes, we hypothesized that the degree of rescue
provided by ionic manganese might be significantly
more than that provided by copper and iron if it
achieved the required concentration and intracellular
location and had the correct ligands in vivo. If so, we
expected it to at least partially rescue several more of the
well-characterized defects of sod1D yeast than those that
had already been discovered [8, 17], and that these data
might provide us with valuable clues as to its mechanism
of protective action. We expected the manganese-con-
taining SOD mimetic compounds to provide a signifi-
cant degree of rescue to the sod1D yeast as well.
Our results confirmed that ionic manganese at high
levels is beneficial to sod1D yeast [8, 17]; however, the
extent of the rescue surprised us. We found that addition
of exogenous manganous ion to the growth medium at
millimolar levels fully rescued all known growth-related
phenotypes of yeast lacking CuZnSOD. In other words,
we found no significant differences between the pheno-
types of sod1D yeast grown in medium containing 5 mM
manganous ions and wild-type yeast. In addition, we
were surprised to find that the manganese-containing
SOD mimetic compounds had no beneficial effect at all
and instead retarded growth of the sod1D yeast. These
results suggest the possibility that non-SOD manganese
may play an antioxidant role in some eukaryotic
organisms.
Materials and methods
Reagents, media, and cell growth
The S. cerevisiae yeast strain EG103 (MATa leu2-3 112
his3D1 trp-289a ura3-52) and the isogenic sod1D strain
derived from it, EG118 (MATa leu2-3, 112 his3D1 trp-
289a ura3-52 sod1D::URA3) [34] were used in this

study. MnCl2Æ4H2O and ZnCl2 were purchased from
Fisher. Unless otherwise noted, the yeast cells were
cultured in synthetic complete medium with 2% glu-
cose (SDC) composed as described in Ref. [35], with
the exceptions that the supplements Leu, His, Trp,
Met, Ura, and Ade were increased fourfold and the pH
was adjusted to 4.0 or 6.0 as indicated. Overnight
startup cultures were grown from single colonies for all
experiments. Yeast strains were inoculated at a starting
OD600 of 0.05 or 0.1 (approximately 0.5-1·106 cells/
mL) in Erlenmeyer flasks with a liquid-to-flask volume
ratio of 1:5. Cultures were incubated at 30C, with
shaking at 220 rpm in air unless otherwise noted. For
indicated experiments, cultures were inoculated and
grown as described above except under an atmosphere
of 100% dioxygen.
Enzyme activity assays
For isopropylmalate dehydratase (Leu1p) and aconi-
tase assays, 5·108 cells resuspended in 0.5 mL of
standard lysis buffer were subjected to glass bead lysis
performed under nitrogen in a septum-sealed glass test
tube. Two-hundred microliters of 0.5-mm glass beads
with six cycles of 30 s vortexing followed by 30 s on
ice was used. Cell debris was removed by centrifuga-
tion at 4,000 rpm (3,080g) for 5 min, and the super-
natant was assayed for protein concentration by
Bradford assay (BioRad). Leu1p activity was deter-
mined spectrophotometrically by monitoring the dis-
appearance of citraconate (Aldrich) at 235 nm as
described in Ref. [36]. Briefly, a 0.5-mL assay mixture
containing 4 mM potassium phosphate, pH 7.0, 0.4
mM citraconate, and 100-300 lg of lysate protein was
assayed for 3 min in a 2-mm path length cuvette [37].
Aconitase activity was determined spectrophotometri-
cally as described by Gardner et al. [38]. The assay
mixture contained 50 mM tris(hydroxymethyl)amino-
methane (Tris)-HCl, pH 7.5, 5 mM sodium citrate, 0.6
mM MnCl2, 0.2 mM NADP+, 2 units of NADP+
isocitrate dehydrogenase, and 50-100 lg of protein.
The appearance of NADPH was monitored at 340 nm
for 5 min. For SOD activity assays, samples were
prepared as just described, but not under nitrogen.
SOD activity measurements were made using activity
gels, as described in Ref. [39].
Stationary-phase survival
Yeast cells were inoculated and incubated as described
before for 72 h. In order to monitor cell viability in the
stationary phase, a serial dilution series was performed
and samples were spotted on YPD plates (1% yeast
extract, 2% peptone, 2% dextrose, and 2% agar) and
incubated at 30C in a low dioxygen environment
(roughly 5% dioxygen, Campy Bags, Becton Dickinson
Microbiology Systems) for 3 days.
915
Visualization of vacuoles
The fluorescent dye FM-4-64 (Molecular Probes), which
specifically stains vacuolar membranes, was used. After
the cells had reached the log phase (OD600<1.5) in SDC
pH 4.0 with or without 5 mM Mn2+ added, approxi-
mately 2.5·107 cells were harvested and incubated aer-
obically with 50 lM FM-4-64 in SDC pH 4.0 (without
any added manganese) for 15 min. The cells were then
resuspended in SDC pH 4.0 with or without added
manganese and grown for at least 1 h prior to visuali-
zation using a Zeiss Axioplan 2 microscope. Images were
recorded using a Zeiss digital camera.
EPR sample preparation
EPR samples were prepared as described in Ref. [40].
After 72 h of growth, 10-20 mL of culture was spun
down at 4,000 rpm for 10 min at 4C. The cell pellet
(approximately 109 cells) was washed once with 10 mL
of cold 20 mM Tris-Cl, pH 7.4, resuspended in 200 lL of
20 mM Tris-Cl, pH 7.4, containing 10% glycerol, and
transferred into an EPR tube. The sample was frozen in
dry ice and stored at -70C until EPR measurements
were performed.
Whole-cell low-temperature Fe(III) EPR
Spectra were recorded with a Bruker X-band spec-
trometer. Samples were maintained below -178C during
the recording of the spectra either using a finger Dewar
filled with liquid nitrogen or a variable-temperature gas-
cooled cavity. The parameters for low-temperature
Fe(III) EPR using the finger Dewar were as reported in
Srinivasan et al. [14]. The parameters for EPR using the
variable-temperature cavity were as follows: center field,
1,560 G; sweep width, 500 G; frequency 9.45 GHz;
microwave power, 31 mW; attenuation, 10 dB; modu-
lation amplitude, 20 G; modulation frequency, 100 kHz;
receiver gain, 2·105; sweep time, 20.97 s; time constant,
81.92 ms; resolution, 2,048 points; number of scans, 16.
EPR data processing was carried out using the Bruker
WinEPR program. Quantitation and calculation of
EPR-detectable iron levels were carried out as described
previously [40].
Measurement of total copper, zinc, iron, and manganese
Triplicate samples, each containing approximately
3.5·108 cells (35 OD600 units) per sample, were collected
from 50-mL cultures (grown for either 24 or 72 h) by
centrifugation (5 min at 4,000 rpm). Samples were wa-
shed twice in 4 mL of 10 mM EDTA, pH 8, and twice in 1
mL of high-purity water (VWR Scientific). The cell pellet
was resuspended in 1 mL of 20% ultrapure nitric acid
(Fisher) and heated at 90-95C for at least 18 h. After
916
complete digestion, 0.9 mL of the sample was diluted to
4.0 mL with high-purity water and analyzed using a
Thermo-Jarrel Ash Iris 1000 inductively coupled plasma
atomic emission spectrometer (ICP-AES). The intracel-
lular yeast volume of 70 lm3 [41] and the number of cells
used for sample preparation were used to quantitate
metal levels inside yeast cells for values reported as
concentrations. Otherwise, metal levels are reported as
parts per billion per OD600 (approximately 107 cells).
Manganese-containing SOD-mimetic compounds
The manganese salen derivatives, Euk-8, Euk- 134, and
Euk-189, were provided by Susan R. Doctrow (Eukari-
on, Bedford, MA, USA). The SOD-active and SOD-
inactive manganese cyclam derivatives, M40403 and
M40404, respectively, were provided by Dennis P. Riley
(Metaphore Pharmaceuticals, Fort Lee, NJ, USA). See
Structure 1 for structures. Euk-8, Euk-134, and Euk-189
were dissolved in water at 10 mM and M40403 and
M40404 at 1.0 mM. Both were filter-sterilized prior to
addition to the growth medium at the indicated con-
centrations.
Results
We tested a wide variety of phenotypes that are evi-
denced in sod1D yeast, and 5 mM ionic manganese fully
reversed the phenotype in every case. Five millimolar
Mn2+ was chosen because it was the concentration at
which the maximum rescue of the sod1D strain was ob-
a in the presence of high dioxygen in either carbon source.
N N
Mn
O Cl O strain and a similarly sized decrease in growth for the
R R
Compound
EUK-8
EUK-134
EUK-189
b a lower 24-h optical density when glycerol was given as
N N
Cl
N
N
Mn
N
N Cl N N
M40403 M40404
Structure 1
served (data not shown). Lower concentrations were not
as effective, and higher ones were toxic. In fact, 5 mM is
borderline toxic; it can be noted in most of the figures
that the wild-type yeast was slightly impaired by high
manganese treatment.
Growth in air or 100% O2
It was previously reported that the addition of milli-
molar concentrations of Mn2+ to the growth medium
rescued the aerobic methionine auxotrophy and growth
on rich plates in 100% dioxygen of sod1D yeast [8].
Lapinskas et al. [17] demonstrated that Mn2+ rescued
the lysine auxotrophy of the sod1Dsod2D double mutant
lacking both CuZnSOD and mitochondrial MnSOD. It
was not clear whether this rescue would extend to all
other observed phenotypes of the sod1D mutant.
Initially, we confirmed that the reported rescue with
Mn2+ could be reproduced in the strain most commonly
used in our laboratory (EG118). Using defined medium
lacking either lysine or methionine (SD-Lys or SD-Met,
respectively), we confirmed that the lysine and methio-
nine auxotrophies, characteristic of sod1D yeast grown
in liquid medium in air, were rescued similarly regardless
of whether the initial medium pH was 4.0 or 6.0 and that
both MnCl2 and MnSO4 salts were equally effective
(data not shown).
To study whether the addition of exogenous Mn2+
would improve the growth of the sod1D strain, EG118,
we examined growth under normal atmospheric and
100% dioxygen conditions in glucose and in the non-
fermentable carbon source glycerol. Wild-type growth
was unaffected by the increase in dioxygen concentra-
tion, whereas sod1D strain growth was severely restricted
In air, sod1D yeast grows to a lower saturation density
than wild-type yeast. Addition of 5 mM MnCl2 effected
a modest improvement in growth (20%) for the sod1D
wild-type strain, such that their final densities were the
same (Fig. 1a). The effect of Mn2+ on sod1D yeast
grown in 100% O2 was more striking. The addition of 5
R group mM MnCl2 to SDC medium afforded full protection
H against the increase in O2 assault that is intolerable to
OCH3 the mutant in unsupplemented medium (Fig. 1b). Again,
OCH2CH3 5 mM Mn2+ caused a small decrease in growth in the
wild-type strain. Both wild-type and sod1D yeast grew to
the carbon source. When 5 mM MnCl2 was added to
SGC medium, there was no significant effect on wild-
Cl
type yeast, while sod1D yeast was again rescued to wild-
Mn
N
type levels (Fig. 1c).
Cl N
Temperature sensitivity
Wild-type yeast grew to a 30% lower density when
grown at 37C for 24 h than it did when grown at 30C
 917

a 8 addition of exogenous Mn2+ provided a dramatic

benefit to the sod1D yeast. The growth at 37C with 5

Growth (OD at 600 nm)

Air
mM Mn2+ was 11 times that of sod1D yeast incubated
6 without added manganese (Fig. 2a), and this was very
close to the growth seen in wild-type yeast with added
4 manganese.

2
Stationary-phase survival
0
wild type sod1∆ A previous report showed that another characteristic of
sod1D yeast is that they do not survive as well as wild-
b 8 type yeast in the stationary phase [34]. Wild-type and
sod1D yeasts were grown for 72 h (to the stationary
Growth (OD at 600 nm)

100% O2
6 phase) with shaking at 220 rpm in air in SDC medium at
30C. Five microliters of a tenfold serial dilution series
4 of these yeast were spotted onto rich medium plates and
grown in a low dioxygen environment (Fig. 2b). As was
previously observed [42], wild-type yeast survival after
2
this incubation far exceeded the survival of sod1D yeast
(Fig. 2b). Again, we saw a significant improvement when
0 the mutant yeast was incubated with 5 mM MnCl2 in
wild type sod1∆ liquid culture and a slightly diminished growth of the
wild-type yeast, such that wild-type and sod1D yeast
c
Growth (OD at 600 nm)

0.8 SGC

0.6

0.4

0.2

0
wild type sod1∆

Fig. 1 Mn2+ enhanced growth of yeast lacking the copper-zinc

superoxide dismutase gene (sod1D) in air and in a 100% O2
atmosphere. Wild-type EG103 and isogenic sod1D EG118 strains
were seeded at OD600 of 0.05 in minimal medium in the absence
(white bars) or presence (black bars) of 5 mM MnCl2. a Total
growth following 24 h of shaking in synthetic complete medium
with 2% glucose (SDC) at 220 rpm in air at 30C was measured
turbidimetrically at 600 nm (OD600). b Total growth (OD600)
measured after incubation under identical growth conditions in a
100% O2 atmosphere. c Total growth (OD600) measured after
incubation in the nonfermentable carbon source, SGC, and in a
100% O2 atmosphere. The values represent the average of two or
more independent samples grown on the same day, with error bars
indicating the standard deviation between samples. The experiment
was repeated at least three times and the data are representative of
these trials
(data not shown). By contrast, sod1D yeast exhibited a
much more dramatic decrease in density after growth at
37C, reaching an OD600 only 4% of that observed after
the normal 30C incubation (data not shown).
Ionic manganese (5 mM MnCl2) was added to the
cultures to test for rescue of this phenotype. In the case
of wild-type yeast, the additional Mn2+ further re-
duced the growth at 37C (Fig. 2a). However, the
Fig. 2 Added Mn2+ affected growth of wild-type and sod1D yeast
incubated at elevated temperatures and extended survival of sod1D
in the stationary phase. a The indicated strains were grown in SDC
medium in the absence (white bars) or presence (black bars) of 5
mM MnCl2. Total growth following a 24-h growth period, with
shaking at 220 rpm in air at 37C, was measured turbidimetrically
at 600 nm (OD600). The values represent the average of three
independent samples grown on the same day, with error bars
indicating the standard deviation between samples. The experiment
was repeated at least three times and the data are representative of
these trials. b Wild-type EG103 and isogenic sod1D (EG118) strains
were incubated in SDC in the absence or presence of 5 mM MnCl2
for 72 h. Cells were viability-tested by spotting 5·104, 5·103, 5·102,
and 5·101 cells onto YPD plates (1% yeast extract, 2% peptone,
2% dextrose, and 2% agar) and incubated at 30C in a low
dioxygen environment. A characteristic result is shown. The
experiment was repeated at least three times using three indepen-
dent colonies and the same trend was observed
918

survived to the same degree. Thus, we concluded that the
defect in stationary-phase survival characteristic of
sod1D yeast at day 3 was also rescued by exogenous
manganese.
[4Fe-4S] cluster enzyme activity
The best known targets of in vivo superoxide toxicity are
enzymes that contain a [4Fe-4S] cluster with a labile,
solvent-exposed iron atom specifically susceptible to
superoxide inactivation [43]. Yeasts contain at least
three such enzymes-aconitase, Leu1p, and homoaconi-
tase-all of which exhibit decreased activity in sod1D cells
[37]. Leu1p is cytoplasmic, like CuZnSOD, and aconi-
tase is in the mitochondrial matrix. Supplementation of
the growth medium with 5 mM MnCl2 resulted in a
significant increase in the activity of Leu1p in sod1D
cells, while the wild-type cells show slightly diminished
two strains after 72 h of growth in the presence and ab-
sence of 5 mM MnCl2. Growth with Mn2+ resulted in a
more than 60% decrease in EPR-detectable iron accu-
mulation in the sod1D mutant relative to growth without
added manganese. Conversely, wild-type yeast accumu-
lated approximately 50% more FeIII g=4.3 when grown in
the presence of 5 mM Mn2+ (Fig. 4a). Thus, after growth
with manganese, we observed only a twofold elevation of
FeIII
g=4.3 in sod1D yeast over wild-type yeast.
We also measured the total iron accumulated by the
two strains after growth under identical conditions. In
contrast to the EPR-detectable iron, the total iron did
not change dramatically with the addition of manganese.
Without 5 mM Mn2+, the amounts of total iron
amassed by wild-type and sod1D yeasts were statistically
indistinguishable. With 5 mM Mn2+, both strains
a 0.12

(∆A/min/mg protein)
activity (Fig. 3a). A similar pattern was observed for 0.01

Leu1p Activity
aconitase (Fig. 3b) though it was not as dramatic.
0.08
0.06
Vacuolar fragmentation 0.04
0.02
When yeasts are grown under normal conditions, the 0
majority of wild-type cells have one or two large vacu- wild type sod1∆
oles per cell. This type of vacuolar morphology has been
termed type A, whereas a pattern of three or more b 50
smaller vacuoles has been termed type B vacuolar
(mU/mg protein)

40
Aco1p Activity

morphology [44]. Corson et al. [45] reported that yeast

30
lacking CuZnSOD exhibited increased vacuole frag-
mentation compared with wild-type yeast when grown 20
aerobically. In our wild-type strain grown in SDC at pH
10
4.0 and treated with FM-4-64 to visualize the vacuolar
membranes, less than 25% of the wild-type population 0
wild type sod1∆
contained type B vacuoles. In contrast, sod1D yeast
grown in the same conditions showed apparent vacuolar
fragmentation-more than two thirds of the cells had c 100
Vacuolar Morphology

three or more smaller vacuoles, i.e., exhibited type B type A

(percent of cells)

morphology (Fig. 3c). When sod1D yeast was grown in
the presence of 5 mM Mn2+, the fraction of sod1D cells
exhibiting type B morphology decreased to 36% (Fig.
3c). These values are similar to those observed for the
wild-type control and suggest that the addition of
exogenous manganese ions also rescues this phenotype.
In concert with previous findings suggesting that high
levels of manganese are somewhat detrimental to wild-
type yeast, 5 mM manganese added to the medium re-
sulted in a slight increase in vacuolar fragmentation in
wild-type yeast (data not shown).
EPR-detectable (free) iron and total iron levels
The amount of ferric iron detectable by EPR at g=4.3
(FeIII
g=4.3) is roughly 5 times greater in EG118 yeast
(lacking CuZnSOD) than in its wild-type parent strain,
EG103 [14]. We measured the amount of FeIII
g=4.3 in these
80
type B
60
40
20
0
wild type sod1∆ sod1∆+Mn
Fig. 3 Growth in the presence of added Mn2+ results in sod1D
yeast with Leu1p activity, Aco1p activity, and vacuole morphology
more similar to those of wild-type cells. a, b The indicated strains
were grown in SDC medium in the absence (white bars) or presence
(black bars) of 5 mM MnCl2. a Leu1p activity. b Aco1p activity.
The average of data from four or more colonies is reported. c
Vacuolar morphology of wild-type and sod1D yeasts was visualized
after growth with or without 5 mM MnCl2 followed by growth
with 50 lM FM-4-64 as described in ‘‘Materials and methods.’’
Type A vacuolar morphology refers to cells with one to two
vacuoles, which is considered normal, whereas a pattern with three
or more smaller vacuoles has been termed type B [44]. The average
of at least 400 cells from six colonies (three separate fields per
colony) is shown

a in the presence and absence of 5 mM MnCl2. While
100
EPR-Detectable Iron (µM)
80
60
40
20
0 sensitivity of sod1D yeast, as the mutant yeast reached the
wild type sod1∆
b 300
Total Iron (µM)
200
100
0 wild-type and sod1D yeasts grown for 24 h in SDC with
wild type sod1∆
Fig. 4 Growth in the presence of exogenous Mn2+ resulted in
decreased levels of electron paramagnetic resonance (EPR)
detectable iron in sod1D yeast while total iron levels remained
relatively unchanged. The indicated strains were seeded at OD600 of
0.05 in minimal medium in the absence (white bars) or presence
(black bars) of 5 mM MnCl2. Iron was measured after a 72-h
incubation period. a Low-temperature iron EPR was employed to
measure the levels of FeIII
g=4.3 accumulation. b Total iron levels were
measured using a inductively coupled plasma atomic emission
spectrometer (ICP-AES) and are reported as the concentration of
iron. The values represent the average of two or more independent
samples, with error bars indicating the standard deviation between
samples
accumulated on average about 10% more total iron, but
the difference was not statistically significant (Fig. 4b).
Accumulation of Mn2+
We determined the intracellular concentration of Mn2+
in wild-type and sod1D yeasts in order to determine
whether a specific intracellular level of this metal was
required for the observed rescue. We used an ICP-AES to
measure the manganese content in cells after growth
under our standard conditions (SDC pH 4.0, shaking in
air at 30C for 24 h) with or without the addition of 5
mM MnCl2. When no extra manganese was added, the
sod1D yeast accumulated similar levels of manganese to
wild-type cells. When yeast cells were grown on minimal
medium supplemented with 5 mM MnCl2, both wild-
type and sod1D yeasts acquired high levels of manganese
(Fig. 7).
Hypersensitivity to zinc
Wei et al. [15] demonstrated that 2 mM Zn2+ inhibited
the growth of sod1D yeast. This experiment was repeated
919
the growth of wild-type yeast after 24 h was unaffected by
the presence of 2 mM ZnCl2 in the growth medium, the
sod1D yeast mutant exhibited a dramatic growth defect
[15]. The addition of 5 mM MnCl2 to growth medium
supplemented with 2 mM ZnCl2 had a small growth
inhibitory effect on wild-type yeast and a dramatic stim-
ulatory effect on sod1D yeast (90% growth enhancement).
The extra Mn2+ resulted in complete rescue of the zinc
same OD600 as the wild-type yeast (Fig. 5).
Accumulation of zinc
We considered it possible that Mn2+ rescue results from
competition for binding and transport, resulting in a
reduction of the amount of zinc entering the cell. To
address this question, the zinc content was analyzed in
or without 5 mM MnCl2, in the presence or absence of 1
mM ZnCl2. In these experiments, 1 mM ZnCl2 was used
because the sod1D cells, although growth-inhibited, still
grew enough at this zinc concentration to provide suf-
ficient sample for metal analysis. Without added zinc,
sod1D yeast accumulated almost twice as much zinc as
wild-type yeast. However, in the presence of exogenous
manganese, the amount of zinc in the two strains was the
same (Fig. 6a). In SDC with 1 mM ZnCl2, sod1D yeast
amassed approximately 25% more zinc. Supplementa-
tion of SDC with 5 mM MnCl2 in addition to 1 mM
Zn2+ resulted in a decrease in zinc accumulation in both
strains, again restoring wild-type zinc levels in the sod1D
mutant (Fig. 6b).
Effect of Zn2+ on manganese accumulation
To test whether zinc toxicity is related to an effect of zinc
on intracellular manganese levels, the total manganese
10
Growth (OD at 600 nm)
SDC
8
SDC+5mM Mn
6
4
2
0
- Zn + Zn - Zn + Zn
wild type sod1D
Fig. 5 Mn2+ rescued the hypersensitivity of the sod1D yeast to
millimolar Zn2+ concentrations. The indicated strains were seeded
at OD600 of 0.05 in minimal medium in the absence (white bars) or
presence (black bars) of 5 mM MnCl2 and with or without 2 mM
ZnCl2 added as indicated. Total growth following a 24-h
incubation period was measured turbidimetrically at 600 nm. This
is an average of six colonies grown on two different days, with error
bars indicating the standard deviation between colonies
920
a result in a further augmentation. Indeed, if anything, the
Total Zn (ppb/OD) 7 total MnSOD activity decreased in the manganese-
6 SDC
5 SDC +5mM Mn
4
3
2
1
0
wild type sod1D
b 200
SDC +1mM Zn
Total Zn (ppb/OD)
160 SDC +1mM Zn +5mM Mn
120
80
40
0 mimics to determine their effectiveness in our yeast
wild type sod1D
Fig. 6 Effect of manganese on zinc accumulation in wild-type and
sod1D yeasts. a Wild-type EG103 and isogenic sod1D yeasts were
grown for 24 h in SDC (white bars) or SDC plus 5 mM MnCl2
(black bars). b The same strains were grown for 24 h in SDC with 1
mM ZnCl2 (gray bars) or in SDC with both 1 mM ZnCl2 and 5 mM
MnCl2 (white bars). In both experiments, cells were collected,
washed, and total zinc was measured with an ICP-AES and is
reported as the amount of zinc per optical density (approximately
107 cells). The values are the average of at least 12 samples,
harvested and analyzed on two different days. Error bars represent
the standard deviation
content was measured in wild-type and sod1D strains
after growth in SDC supplemented with 5 mM MnCl2, 1
mM ZnCl2, or both. Growth in SDC plus 1 mM ZnCl2
resulted in a reduction in manganese in wild-type and
sod1D yeasts by about 33% when compared with yeast
grown in SDC without added metals (Fig. 7a). This
influence of zinc on the total manganese content was
similar for wild-type and sod1D yeasts grown in SDC
plus 5 mM MnCl2; the strains accumulated about 20%
less manganese when zinc was present in addition to the
excess manganese (Fig. 7b).
MnSOD activity
One possible explanation for our results could be that
excess manganese functions by activating the endoge-
nous MnSOD, which is still present in the mitochondrial
matrix of the sod1D strains. Two pieces of evidence ar-
gue against this possibility. First, manganese rescue is
equally effective in an sod1Dsod2D double knockout
strain: MnCl2 fully rescues the lysine auxotrophy [17],
hypersensitivity to elevated temperature, and hypersen-
sitivity to ZnCl2 of the sod1Dsod2D double knockout
strain (data not shown). Second, we performed gel-based
SOD activity assays on wild-type and sod1D strains (Fig.
8). Although the sod1D strain appears to have somewhat
more MnSOD activity than the SOD1+ strain, the
addition of manganese to the growth medium did not
treated samples.
Growth in the presence of manganese-containing
SOD mimics
A number of manganese-containing coordination com-
plexes, studied in cell culture and whole animals, were
shown to be effective in preventing damage attributed to
oxidative stress [20, 21, 46] (see ‘‘Introduction’’). On this
basis, it has been assumed that these compounds are
acting as functional SOD mimics. To our knowledge, the
effect of manganese-containing SOD mimics has not
been studied previously in a yeast model system. We
therefore examined several manganese-containing SOD
model system.
The Mn(III) salen derivatives from Eukarion are a
unique class of antioxidants that exhibit both SOD
activity and catalase activity. We tested Euk-8, Euk-134,
and Euk-189, which differ in the nature of the substit-
uents on the salen moiety. They were reported to have
the same level of SOD activity but differed in their levels
of catalase activity and lipophilicity. The catalase
activity of the prototypical Euk-8 (148 lM O2/min) was
reported to be lower than that of Euk-134 or Euk-189
(243 and 180 lM O2/min, respectively), and Euk-189
a 0.4
Total Mn (ppb/OD)
SDC
0.3
SDC +1mM Zn
0.2
0.1
0
wild type sod1D
b 20 SDC +5mM Mn
Total Mn (ppb/OD)
16 SDC +1mM Zn +5mM Mn
12
8
4
0
wild type sod1D
Fig. 7 Effect of zinc on manganese accumulation in wild-type and
sod1D yeasts. a Wild-type EG103 and isogenic sod1D yeasts were
grown for 24 h in SDC (white bars) or SDC plus 1 mM ZnCl2 (gray
bars). b The same strains were grown for 24 h in SDC with 5 mM
MnCl2 (black bars) or in SDC with both 1 mM ZnCl2 and 5 mM
MnCl2 (white bars). In both experiments, cells were collected,
washed, and the total manganese was measured with an ICP-AES
and is reported as the amount of manganese per optical density
(approximately 107 cells). The values are the average of at least 12
samples harvested and analyzed on two different days. Error bars
represent the standard deviation

Fig. 8 SOD activity. Native polyacrylamide gel electrophoresis of
yeast extracts, showing endogenous SOD enzyme activity deter-
mined by in-gel activity assay a after and b before cyanide
was found to be more lipophilic than either Euk-8 or
Euk-134 [20].
The aerobic growth of wild-type and sod1D S. cere-
visiae cultured in SDC pH 6.0 in the presence of each of
the Eukarion compounds was recorded after 24 h. Wild-
type yeast cells were largely unaffected by treatment with
Euk-8, Euk-134, or Euk-189. The poor aerobic growth
of sod1D yeast was not improved upon treatment with
any concentration of Euk-8, Euk-134, or Euk-189 tested,
in stark contrast to the rescue observed with ionic
Mn2+. Instead, relatively low concentrations of these
compounds prevented growth of these strains. We
interpret this effect to mean that the compounds are able
to enter the cells.
In order to determine whether any beneficial effects of
the Eukarion compounds might be observed under more
stringent conditions, the 24-h aerobic growth of wild-
type and sod1D yeast in SDC pH 6.0 lacking lysine or
methionine was monitored in the presence of up to 3 lM
Euk-134. Wild-type yeast was not affected by the pres-
ence of Euk-134 in either drop-out medium. Very little
growth of sod1D S. cerevisiae in either SD-Lys or SD-
Met was observed, and the growth was not improved
with the addition of Euk-134 (data not shown).
The Mn(III) cyclam derivative M40403 from Meta-
phor that we tested has been reported to be highly sta-
ble, with a high level of SOD activity and no catalase
activity [21, 47]. Aerobic growth of wild-type S. cerevi-
siae in the presence of M40403 remained unchanged
from the untreated control as measured after 24 h (Fig.
9b). sod1D yeast cells cultured with M40403 were not
rescued by low levels of this mimic (0.05 and 5 lM, data
not shown), and, in fact, their growth was inhibited by
about 90% with 25 lM M40403 (Fig. 9b). The struc-
turally related SOD-inactive control compound M40404
had no effect on wild-type yeast or mutant yeast at the
same concentrations. The observation of growth inhi-
bition for sod1D yeast cultured with M40403 is strong
evidence that this manganese complex enters yeast cells;
on the basis of their structural similarities, it seems likely
that M40404 enters yeast cells as well.
Discussion
Our studies demonstrate that the beneficial effects of
manganese supplementation on sod1D yeast are much
more dramatic and complete than those of copper or
921
treatment to inhibit the CuZnSOD. The samples are wild-type
yeast (1), wild-type yeast treated with 5 mM MnCl2 (2), sod1D yeast
(3), and sod1D yeast treated with 5 mM MnCl2 (4)
iron: all of the known sod1D yeast growth phenotypes
were rescued fully, including the zinc sensitivity and the
iron-related defects such as reduced aconitase and Leu1p
activities and elevated levels of EPR-detectable iron.
There are some systems in which the effect of elevated
levels of manganese may be attributed to an increase in
the activity of the mitochondrial MnSOD [48]. We have
found that this is not the case in yeast. First, we con-
firmed the earlier observation that Mn2+ rescues the
sod1Dsod2D double mutant, which lacks MnSOD [17].
Second, activity gels showed no increase in activity of
MnSOD in cells treated with high manganese (Fig. 8). If
anything, the activity of MnSOD was decreased by high
manganese in both wild-type yeast and the sod1D mu-
tant. S. cerevisiae normally requires only low levels (less
than 3 lM) of manganese in the medium to sustain
a
10
Growth (OD at 600 nm)
WT Euk-134
8 sod1∆ Euk-134
6 WT Euk-8
sod1∆ Euk-8
4
WT Euk-189
2 sod1∆ Euk-189
0
1 10 100
Eukarion Concentration (µM)
b
Growth (OD at 600 nm)
10
8
SDC
active (M40403)
6 inactive (M40404)
4
2
0
wild type sod1∆
Fig. 9 The presence of Eukarion or Metaphore SOD mimics does
not improve growth of sod1D Saccharomyces cerevisiae. Total
growth following 24 h of shaking in SDC pH 6.0 at 220 rpm in air
at 30C was measured turbidimetrically at 600 nm. a Growth of
wild-type EG103 yeast (solid symbols) and isogenic sod1D yeast
(open symbols) with various concentration of Euk-134 (squares),
Euk-8 (triangles), or Euk-189 (circles). b Growth of the same strains
in the presence of 25 lM active Metaphore compound M40403
(black bars), the inactive Metaphore control M40404 (gray bars), or
the untreated control (white bars). The values represent the average
of six or more independent colonies grown on two or more days,
with error bars indicating the standard deviation
922
growth. It is interesting to note that in our experiments
the concentration of MnCl2 that provided the optimal
rescue of the sod1D yeast, i.e., 5 mM, was the same
concentration at which a small but significant negative
effect is first observed on control wild-type cells. Below
that concentration, the addition of MnCl2 to wild-type
cells had no effect on growth, and extremely high levels
of manganese (8-10 mM) were toxic. One possible
explanation is that 5 mM is the lowest concentration of
Mn2+ in the medium that provides a rate of influx of
Mn2+ that is not totally counteracted by the homeo-
static mechanisms in place to handle toxic levels of
exogenous metals.
The rescue by manganese of the zinc sensitivity of the
sod1D yeast was particularly striking to us, since it indi-
cates that the absence of the SOD1 polypeptide does not
result in zinc sensitivity so long as the cell can otherwise
totally defend itself against superoxide. Mn2+ does not
appear to be competing with Zn2+ for import because
intracellular zinc levels are not lowered when cells are
grown in 5 mM MnCl2 (Fig. 7b). Thus, although the
possibility remains that manganese displaces zinc from
some important site that mediates zinc toxicity without
changing cellular zinc levels, the present data appear to
support the hypothesis that Mn2+ rescue of sod1D yeast
is due primarily to its antioxidant action in vivo. In our
previous study [15], zinc resistance was not restored by
cytosolic expression of MnSOD from Bacillus stearo-
thermophilus; this bacterial enzyme did however rescue
paraquat sensitivity [15]. This result seems contradictory
to our current conclusion that Mn2+ rescue of sod1D
yeast is due to its ability to scavenge superoxide. One
possible explanation is that Mn2+ may be capable of
protecting in a subcellular compartment not accessible to
the bacterial MnSOD, a compartment that might be
particularly sensitive to the adverse effects of high zinc. A
likely candidate is the IMS, for the following reasons.
Zinc and manganese can easily access this space via pores
in the outer membrane that permit the passage of small
water-soluble molecules, but proteins (i.e., the bacterial
MnSOD) and other large molecules that are not sub-
strates for specific transport across the membrane are
excluded. Superoxide is produced in the IMS, CuZnSOD
is ordinarily present, and CuZnSOD in this area was
reported to be important for long-term survival of yeasts
[42]. High zinc is detrimental to many mitochondrial
functions [49]. If this compartment is especially sensitive
to high levels of superoxide, then in the absence of active
CuZnSOD it could be protected either by correct zinc
handling, insured by the zinc-binding function of inactive
SOD1p (in yeast lacking the CCS1 (LYS7) gene,1ccs1D,
or sod1D yeast expressing H46C-SOD, an SOD-inactive,
zinc-binding mutant of SOD1), or by the superoxide
1
This gene was first identified as part of the lysine biosynthetic
pathway, hence the name LYS7. Since then, its true function-
insertion of copper into CuZnSOD-has been discovered. We sup-
port a recent proposal to rename the yeast gene CCS1 to better
reflect its function and to agree with the names of similar genes in
other organisms.
scavenging ability of Mn2+ present in the compartment.
We find that manganese does accumulate to high levels in
mitochondria of cells grown in high manganese (data not
shown), lending support to this hypothesis.
The deleterious effect of the two classes of manga-
nese-containing SOD mimics, the manganese salen
derivatives, Euk-8, Euk-134, and Euk-189, and the
manganese cyclam complex, M40403, on the growth of
the sod1D yeast was quite surprising to us. In the case of
the manganese cyclam derivatives, we have the advan-
tage of being able to compare two very similar coordi-
nation complexes of manganese, one of which has high
SOD activity and one of which does not. The SOD-
active compound, M40403, inhibited the growth of the
sod1D yeast, while the SOD-inactive compound,
M40404, had no effect. The fact that M40403 inhibited
growth is evidence that this compound does enter yeast
cells; the structural similarity of M40403 and M40404
make it likely that the latter compound enters yeast cells
as well. These results suggest that the growth inhibitory
effect of the SOD-active manganese-containing com-
pounds on the sod1D yeast may be related to their SOD
activity. It is possible that alterations in levels of ROS
may lead to the disruption of cell signaling, resulting in
poor growth, as the transcription of many genes is in-
duced by ROS [50, 51], but further work is required to
evaluate this intriguing hypothesis. What is clear now
from this study is that these two classes of manganese-
containing SOD mimetic compounds do not provide any
benefit to the sod1D yeast, while the presence of ionic
Mn2+ provides full rescue.
The antioxidant role of ionic Mn2+ in vivo is thought
to be due to its superoxide scavenging ability, and, as
discussed in the ‘‘Introduction’’, a number of prokary-
otic species are thought to use Mn2+ for exactly this
purpose [1]. It now appears that this phenomenon is not
restricted to prokaryotes and that the simple eukaryote
S. cerevisiae can use manganese in this way as well.
Because Mn2+ rescues all the phenotypes tested, we are
inclined to believe that it works by chemically removing
superoxide. Future studies will aim to get a better
understanding of the subcellular localization and ligand
environment as well as the inorganic chemistry of
manganese that underlies its remarkable antioxidant
properties when it is present at high concentrations in
cells lacking SOD enzymes.
Acknowledgements This work was supported by grant DK46828 to
J.S.V. We also gratefully acknowledge the support of the National
Science Foundation Predoctoral Fellowship (to R.J.S.), the NIH
Chemistry-Biology Interface Predoctoral Training grant (to R.J.S.
and M.A.W.), and the University of California Toxic Substances
Research and Teaching Program, Lead Campus Program in Toxic
Mechanisms (to M.A.W.)
References
1. Horsburgh MJ, Wharton SJ, Karavolos M, Foster SJ (2002)
Trends Microbiol 10:496–501

2. Daly MJ, Gaidamakova EK, Matrosova VY, Vasilenko A,
Zhai M, Venkateswaran A, Hess M, Omelchenko MV, Ko-
standarithes HM, Makarova KS, Wackett LP, Fredrickson JK,
Ghosal D (2004) Science 306:1025–1028
3. Al-Maghrebi M, Fridovich I, Benov L (2002) Arch Biochem
Biophys 402:104–109
4. Archibald FS, Fridovich I (1982) Arch Biochem Biophys
214:452–463
5. Cabelli DE, Bielski BHJ (1984) J Phys Chem 88:6291–6294
6. Weiss RH, Flickinger AG, Rivers WJ, Hardy MM, Aston KW,
Ryan US, Riley DP (1993) J Biol Chem 268:23049–23054
7. Bielski BH, Cabelli DE (1991) Int J Radiat Biol 59:291–319
8. Chang EC, Kosman DJ (1989) J Biol Chem 264:12172–12178
9. Cabelli DE, Bielski BHJ (1984) J Phys Chem 88:3111–3115
10. Jacobsen F, Holcman J, Sehested K (1997) J Phys Chem
101:1324–1328
11. Niviere V, Fontecave M (2004) J Biol Inorg Chem 9:119–123
12. Gralla EB, Valentine JS (1991) J Bacteriol 173:5918–5920
13. Sturtz LA, Culotta VC (2002) Methods Enzymol 349:167–172
14. Srinivasan C, Liba A, Imlay JA, Valentine JS, Gralla EB (2000)
J Biol Chem 275:29187–29192
15. Wei JP, Srinivasan C, Han H, Valentine JS, Gralla EB (2001)
J Biol Chem 276:44798–44803
16. De Freitas JM, Liba A, Meneghini R, Valentine JS, Gralla EB
(2000) J Biol Chem 275:11645–11649
17. Lapinskas PJ, Cunningham KW, Liu XF, Fink GR, Culotta
VC (1995) Mol Cell Biol 15:1382–1388
18. Portnoy ME, Liu XF, Culotta VC (2000) Mol Cell Biol
20:7893–7902
19. Tamai KT, Gralla EB, Ellerby LM, Valentine JS, Thiele DJ
(1993) Proc Natl Acad Sci USA 90:8013–8017
20. Doctrow SR, Huffman K, Marcus CB, Tocco G, Malfroy E,
Adinolfi CA, Kruk H, Baker K, Lazarowych N, Mascarenhas
J, Malfroy B (2002) J Med Chem 45:4549–4558
21. Riley DP (1999) Chem Rev 99:2573–2588
22. Melov S, Ravenscroft J, Malik S, Gill MS, Walker DW,
Clayton PE, Wallace DC, Malfroy B, Doctrow SR, Lithgow
GJ (2000) Science 289:1567–1569
23. Sampayo JN, Olsen A, Lithgow GJ (2003) Aging Cell 2:319–
326
24. Baker K, Marcus CB, Huffman K, Kruk H, Malfroy B, Doc-
trow SR (1998) J Pharmacol Exp Ther 284:215–221
25. Pong K, Doctrow SR, Huffman K, Adinolfi CA, Baudry M
(2001) Exp Neurol 171:84–97
26. Melov S, Doctrow SR, Schneider JA, Haberson J, Patel M,
Coskun PE, Huffman K, Wallace DC, Malfroy B (2001)
J Neurosci 21:8348–8353
923
27. Salvemini D, Wang ZQ, Zweier JL, Samouilov A, Macarthur
H, Misko TP, Currie MG, Cuzzocrea S, Sikorski JA, Riley DP
(1999) Science 286:304–306
28. Shimizu K, Rajapakse N, Horiguchi T, Payne RM, Busija DW
(2003) Neurosci Lett 346:41–44
29. Masini E, Cuzzocrea S, Mazzon E, Marzocca C, Mannaioni
PF, Salvemini D (2002) Br J Pharmacol 136:905–917
30. Wang ZQ, Porreca F, Cuzzocrea S, Galen K, Lightfoot R,
Masini E, Muscoli C, Mollace V, Ndengele M, Ischiropoulos
H, Salvemini D (2004) J Pharmacol Exp Ther 309:869–878
31. Udipi K, Ornberg RL, Thurmond KB 2nd, Settle SL, Forster
D, Riley D (2000) J Biomed Mater Res 51:549–560
32. McFadden SL, Ding D, Salvemini D, Salvi RJ (2003) Toxicol
Appl Pharmacol 186:46–54
33. 33.Nicotera TM, Ding D, McFadden SL, Salvemini D, Salvi R
(2004) Audiol Neurootol 9:353–362
34. Longo VD, Gralla EB, Valentine JS (1996) J Biol Chem
271:12275–12280
35. Kaiser C, Michaelis SAM (1994) Methods in yeast genetics.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
New York
36. Kohlhaw GB (1988) Methods Enzymol 166:429–435
37. Wallace MA, Liou LL, Martins J, Clement MH, Bailey S,
Longo VD, Valentine JS, Gralla EB (2004) J Biol Chem
279:32055–32062
38. Gardner PR, Raineri I, Epstein LB, White CW (1995) J Biol
Chem 270:13399–13405
39. Huang TT, Raineri I, Eggerding F, Epstein CJ (2002) Methods
Enzymol 349:191–213
40. Srinivasan C, Gralla EB (2002) Methods Enzymol 349:173-180
41. Sherman F (1991) Methods Enzymol 194:3–21
42. Sturtz LA, Diekert K, Jensen LT, Lill R, Culotta VC (2001)
J Biol Chem 276:38084–38089
43. Beinert H, Holm RH, Munck E (1997) Science 277:653–659
44. Vida TA, Emr SD (1995) J Cell Biol 128:779–792
45. Corson LB, Folmer J, Strain JJ, Culotta VC, Cleveland DW
(1999) J Biol Chem 274:27590–27596
46. Faulkner KM, Liochev SI, Fridovich I (1994) J Biol Chem
269:23471–23476
47. Riley DP, Weiss RH (1994) Abstracts of Papers of the Amer-
ican Chemical Society 208:152-Inor
48. Davis CD, Greger JL (1992) Am J Clin Nutr 55:747–752
49. Cuajungco MP, Lees GJ (1997) Neurobiol Dis 4:137–169
50. Apel K, Hirt H (2004) Annu Rev Plant Biol 55:373–399
51. Azevedo D, Tacnet F, Delaunay A, Rodrigues-Pousada C,
Toledano MB (2003) Free Radic Biol Med 35:889–900