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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- Keywords Manganese Æ Zinc Æ Copper-zinc superoxide
tase (sod1D) exhibit a broad range of phenotypes, many dismutase Æ Oxidative stress Æ Yeast
of which can be rescued by growth in the presence of
high levels of ionic manganese. We undertook a com- Abbreviations EPR: Electron
prehensive survey of the effects of manganese on wild- paramagnetic resonance Æ ICP-AES: Inductively
type and sod1D yeasts and found that 5 mM Mn2+ coupled plasma atomic emission spectrometer Æ IMS:
rescued all known growth-related phenotypes, such as Intermembrane space of mitochondria Æ Leu1p:
slow growth in air, temperature sensitivity, specific Isopropylmalate dehydratase Æ ROS: Reactive oxygen
amino acid auxotrophies, no growth in high oxygen, species Æ SDC: Synthetic complete medium with 2%
poor growth in nonfermentable carbon sources, and glucose Æ SD-Lys: Defined medium lacking lysine Æ
decreased stationary-phase survival. Iron-related phe- SD-Met: Defined medium lacking methionine Æ SOD:
notypes-elevated electron paramagnetic resonance Superoxide dismutase Æ sod1D: Yeast lacking the
detectable (‘‘free’’) iron, decreased aconitase activity, CuZnSOD gene Æ sod2D: Yeast lacking the MnSOD
and fragmenting vacuoles-as well as zinc sensitivity were gene Æ SOR: Superoxide reductase Æ Tris:
also rescued. The activity of manganese superoxide Tris(hydroxymethyl)aminomethane
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, Introduction
addition of two manganese-containing superoxide
dismutase mimetic compounds to the growth medium Non superoxide dismutase (SOD) manganese plays a
did not provide any rescue of sod1D yeast growth but major role in the antioxidant defense mechanisms of
rather had an sod1D-selective inhibitory effect at several prokaryotic organisms [1]. The phenomenon was
micromolar concentrations. Mechanisms by which ionic first noted when bacterial species that lack any detect-
manganese can effect this rescue, while the mimetic able SOD enzymes were found to contain high (milli-
compounds do not, are discussed. 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
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 non-SOD manganese have been shown to work in con-
Department of Chemistry and Biochemistry, UCLA, junction with antioxidant enzymes such as SOD and
607 Charles E. Young Drive, East, catalase as a major component of their defense systems
Los Angeles, CA 90095-1569, USA against reactive oxygen species [1, 2]. High levels of ionic
E-mail: egralla@chem.ucla.edu
Tel.: +1-310-8251946
manganese added to the growth medium also provide a
Fax: +1-310-2069880 substantial degree of rescue of the dioxygen-sensitive
phenotypes of SOD knockout Escherichia coli [3].
C. Srinivasan Æ K. Le The molecular basis for the antioxidant action of
Department of Chemistry and Biochemistry, non-SOD manganese in prokaryotes is unknown. Early
California State University,
Fullerton, CA 92834-9480, USA reports suggested that non-SOD ionic manganese
914

possessed SOD activity, on the basis of the xanthine/ (1) derivatives of Mn(III) salen complexes Euk-8, Euk-
xanthine oxidase/cytochrome c assay [4]. However, fur- 134, and Euk-189 developed by Eukarion [20] and (2)
ther studies determined that ionic manganese interferes derivatives of Mn(III) cyclam complexes M40403 and
with this indirect assay by promoting the oxidation of M40404 developed by Metaphore Pharmaceuticals [21].
reduced cytochrome c [3]. Furthermore, direct assays The Mn(III) salen (Euk) complexes have been reported
using pulse radiolysis or stopped flow techniques have to increase wild-type Caenorhabditis elegans lifespan [22,
established that ionic manganese at physiological pH 23] and were beneficial in other models of oxidative
has little, if any, SOD activity [5-7]. stress, such as ischemia-reperfusion injury in a rat stroke
Despite the fact that ionic manganese lacks signifi- model [24], reactive oxygen species (ROS)-induced
cant superoxide dismutase activity at physiological pH, apoptosis [25], and improved survival of sod2 null mice
there is considerable evidence that extracts from man- [26]. The Mn(III) cyclam derivative M40403 has been
ganese-treated cells nevertheless have a high degree of found to be efficacious in reducing ischemia-reperfusion
superoxide scavenging ability [1, 3, 4, 8]. Manganous ion injury [21, 27-29], beneficial in inflammation models in
is well known to react with superoxide at fast rates in rats [30, 31], and protective of cochlear hair cells [32, 33].
vitro to form a relatively long lived transient complex The present study was undertaken to determine the
[MnO2]+ [5, 7, 9, 10]. If this transient is rapidly reduced nature and degree of the manganese rescue of sod1D
in vivo by an unknown cellular reductant, non-SOD yeast phenotypes and to compare the effect of ionic
manganese may be acting as a superoxide reductase manganese with that of some manganese-containing
(SOR), functioning in a manner analogous to the non- SOD mimics that had earlier been shown to have sig-
heme iron-containing FeSORs [11]. nificant degrees of biological activity in other experi-
The possibility that non-SOD manganese functions as mental systems. On the basis of earlier studies of L.
an antioxidant in vivo has been less explored in eukary- plantarum and other bacteria entirely lacking in SOD
otes than in prokaryotes. Most of the evidence that exists enzymes, we hypothesized that the degree of rescue
in support of such a function comes from studies of provided by ionic manganese might be significantly
Saccharomyces cerevisiae. The budding yeast S. cerevi- more than that provided by copper and iron if it
siae, like most eukaryotes, contains two SODs: CuZn- achieved the required concentration and intracellular
SOD (SOD1) in the cytosol, the intermembrane space of location and had the correct ligands in vivo. If so, we
mitochondria (IMS), and other locations, and MnSOD expected it to at least partially rescue several more of the
(SOD2) in the mitochondrial matrix. Yeast cells lacking well-characterized defects of sod1D yeast than those that
CuZnSOD (sod1D) have severe aerobic growth defects, had already been discovered [8, 17], and that these data
die quickly in the stationary phase, and exhibit lysine, might provide us with valuable clues as to its mechanism
leucine, and methionine auxotrophies under normal of protective action. We expected the manganese-con-
culture conditions [12, 13]. They also exhibit altered iron taining SOD mimetic compounds to provide a signifi-
and zinc metabolism, when grown aerobically, including cant degree of rescue to the sod1D yeast as well.
elevated electron paramagnetic resonance (EPR) detect- Our results confirmed that ionic manganese at high
able iron levels at g=4.3 and zinc sensitivity [14-16]. The levels is beneficial to sod1D yeast [8, 17]; however, the
most common genetic suppressors of CuZnSOD extent of the rescue surprised us. We found that addition
knockout (sod1D) yeast, i.e., pmr1-1 and bsd2-1, lead to of exogenous manganous ion to the growth medium at
dramatic increases in intracellular manganese levels [17, millimolar levels fully rescued all known growth-related
18]. Additionally, the inability of sod1D yeast to survive phenotypes of yeast lacking CuZnSOD. In other words,
on medium lacking lysine or methionine as well as its we found no significant differences between the pheno-
poor growth in 100% dioxygen have previously been types of sod1D yeast grown in medium containing 5 mM
shown to be rescued by the addition of 5 mM MnCl2 to manganous ions and wild-type yeast. In addition, we
the growth medium [8, 17]. were surprised to find that the manganese-containing
Other metal ions, in particular copper and iron, have SOD mimetic compounds had no beneficial effect at all
also been reported to rescue certain phenotypes of sod1D and instead retarded growth of the sod1D yeast. These
yeast when added to the growth medium, but the rescue results suggest the possibility that non-SOD manganese
in each case was incomplete. Copper, working through a may play an antioxidant role in some eukaryotic
mechanism that requires the presence of metallothionein, organisms.
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- Materials and methods
sphere of 100% dioxygen [19]. Similarly, iron supple-
mentation restored growth on glycerol, but not Reagents, media, and cell growth
methionine prototrophy [16]. In addition to simple metal
ions, several classes of manganese coordination com- The S. cerevisiae yeast strain EG103 (MATa leu2-3 112
plexes have been developed as functional SOD mimics his3D1 trp-289a ura3-52) and the isogenic sod1D strain
and tested for biological efficacy in different model sys- derived from it, EG118 (MATa leu2-3, 112 his3D1 trp-
tems. The two of particular interest to us in this study are 289a ura3-52 sod1D::URA3) [34] were used in this
915

study. MnCl2Æ4H2O and ZnCl2 were purchased from Visualization of vacuoles


Fisher. Unless otherwise noted, the yeast cells were
cultured in synthetic complete medium with 2% glu- The fluorescent dye FM-4-64 (Molecular Probes), which
cose (SDC) composed as described in Ref. [35], with specifically stains vacuolar membranes, was used. After
the exceptions that the supplements Leu, His, Trp, the cells had reached the log phase (OD600<1.5) in SDC
Met, Ura, and Ade were increased fourfold and the pH pH 4.0 with or without 5 mM Mn2+ added, approxi-
was adjusted to 4.0 or 6.0 as indicated. Overnight mately 2.5·107 cells were harvested and incubated aer-
startup cultures were grown from single colonies for all obically with 50 lM FM-4-64 in SDC pH 4.0 (without
experiments. Yeast strains were inoculated at a starting any added manganese) for 15 min. The cells were then
OD600 of 0.05 or 0.1 (approximately 0.5-1·106 cells/ resuspended in SDC pH 4.0 with or without added
mL) in Erlenmeyer flasks with a liquid-to-flask volume manganese and grown for at least 1 h prior to visuali-
ratio of 1:5. Cultures were incubated at 30C, with zation using a Zeiss Axioplan 2 microscope. Images were
shaking at 220 rpm in air unless otherwise noted. For recorded using a Zeiss digital camera.
indicated experiments, cultures were inoculated and
grown as described above except under an atmosphere
of 100% dioxygen. EPR sample preparation

EPR samples were prepared as described in Ref. [40].


Enzyme activity assays 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
For isopropylmalate dehydratase (Leu1p) and aconi- (approximately 109 cells) was washed once with 10 mL
tase assays, 5·108 cells resuspended in 0.5 mL of of cold 20 mM Tris-Cl, pH 7.4, resuspended in 200 lL of
standard lysis buffer were subjected to glass bead lysis 20 mM Tris-Cl, pH 7.4, containing 10% glycerol, and
performed under nitrogen in a septum-sealed glass test transferred into an EPR tube. The sample was frozen in
tube. Two-hundred microliters of 0.5-mm glass beads dry ice and stored at -70C until EPR measurements
with six cycles of 30 s vortexing followed by 30 s on were performed.
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 Whole-cell low-temperature Fe(III) EPR
Bradford assay (BioRad). Leu1p activity was deter-
mined spectrophotometrically by monitoring the dis- Spectra were recorded with a Bruker X-band spec-
appearance of citraconate (Aldrich) at 235 nm as trometer. Samples were maintained below -178C during
described in Ref. [36]. Briefly, a 0.5-mL assay mixture the recording of the spectra either using a finger Dewar
containing 4 mM potassium phosphate, pH 7.0, 0.4 filled with liquid nitrogen or a variable-temperature gas-
mM citraconate, and 100-300 lg of lysate protein was cooled cavity. The parameters for low-temperature
assayed for 3 min in a 2-mm path length cuvette [37]. Fe(III) EPR using the finger Dewar were as reported in
Aconitase activity was determined spectrophotometri- Srinivasan et al. [14]. The parameters for EPR using the
cally as described by Gardner et al. [38]. The assay variable-temperature cavity were as follows: center field,
mixture contained 50 mM tris(hydroxymethyl)amino- 1,560 G; sweep width, 500 G; frequency 9.45 GHz;
methane (Tris)-HCl, pH 7.5, 5 mM sodium citrate, 0.6 microwave power, 31 mW; attenuation, 10 dB; modu-
mM MnCl2, 0.2 mM NADP+, 2 units of NADP+ lation amplitude, 20 G; modulation frequency, 100 kHz;
isocitrate dehydrogenase, and 50-100 lg of protein. receiver gain, 2·105; sweep time, 20.97 s; time constant,
The appearance of NADPH was monitored at 340 nm 81.92 ms; resolution, 2,048 points; number of scans, 16.
for 5 min. For SOD activity assays, samples were EPR data processing was carried out using the Bruker
prepared as just described, but not under nitrogen. WinEPR program. Quantitation and calculation of
SOD activity measurements were made using activity EPR-detectable iron levels were carried out as described
gels, as described in Ref. [39]. previously [40].

Stationary-phase survival Measurement of total copper, zinc, iron, and manganese

Yeast cells were inoculated and incubated as described Triplicate samples, each containing approximately
before for 72 h. In order to monitor cell viability in the 3.5·108 cells (35 OD600 units) per sample, were collected
stationary phase, a serial dilution series was performed from 50-mL cultures (grown for either 24 or 72 h) by
and samples were spotted on YPD plates (1% yeast centrifugation (5 min at 4,000 rpm). Samples were wa-
extract, 2% peptone, 2% dextrose, and 2% agar) and shed twice in 4 mL of 10 mM EDTA, pH 8, and twice in 1
incubated at 30C in a low dioxygen environment mL of high-purity water (VWR Scientific). The cell pellet
(roughly 5% dioxygen, Campy Bags, Becton Dickinson was resuspended in 1 mL of 20% ultrapure nitric acid
Microbiology Systems) for 3 days. (Fisher) and heated at 90-95C for at least 18 h. After
916

complete digestion, 0.9 mL of the sample was diluted to served (data not shown). Lower concentrations were not
4.0 mL with high-purity water and analyzed using a as effective, and higher ones were toxic. In fact, 5 mM is
Thermo-Jarrel Ash Iris 1000 inductively coupled plasma borderline toxic; it can be noted in most of the figures
atomic emission spectrometer (ICP-AES). The intracel- that the wild-type yeast was slightly impaired by high
lular yeast volume of 70 lm3 [41] and the number of cells manganese treatment.
used for sample preparation were used to quantitate
metal levels inside yeast cells for values reported as
concentrations. Otherwise, metal levels are reported as Growth in air or 100% O2
parts per billion per OD600 (approximately 107 cells).
It was previously reported that the addition of milli-
molar concentrations of Mn2+ to the growth medium
Manganese-containing SOD-mimetic compounds rescued the aerobic methionine auxotrophy and growth
on rich plates in 100% dioxygen of sod1D yeast [8].
The manganese salen derivatives, Euk-8, Euk- 134, and Lapinskas et al. [17] demonstrated that Mn2+ rescued
Euk-189, were provided by Susan R. Doctrow (Eukari- the lysine auxotrophy of the sod1Dsod2D double mutant
on, Bedford, MA, USA). The SOD-active and SOD- lacking both CuZnSOD and mitochondrial MnSOD. It
inactive manganese cyclam derivatives, M40403 and was not clear whether this rescue would extend to all
M40404, respectively, were provided by Dennis P. Riley other observed phenotypes of the sod1D mutant.
(Metaphore Pharmaceuticals, Fort Lee, NJ, USA). See Initially, we confirmed that the reported rescue with
Structure 1 for structures. Euk-8, Euk-134, and Euk-189 Mn2+ could be reproduced in the strain most commonly
were dissolved in water at 10 mM and M40403 and used in our laboratory (EG118). Using defined medium
M40404 at 1.0 mM. Both were filter-sterilized prior to lacking either lysine or methionine (SD-Lys or SD-Met,
addition to the growth medium at the indicated con- respectively), we confirmed that the lysine and methio-
centrations. 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
Results both MnCl2 and MnSO4 salts were equally effective
(data not shown).
We tested a wide variety of phenotypes that are evi- To study whether the addition of exogenous Mn2+
denced in sod1D yeast, and 5 mM ionic manganese fully would improve the growth of the sod1D strain, EG118,
reversed the phenotype in every case. Five millimolar we examined growth under normal atmospheric and
Mn2+ was chosen because it was the concentration at 100% dioxygen conditions in glucose and in the non-
which the maximum rescue of the sod1D strain was ob- fermentable carbon source glycerol. Wild-type growth
was unaffected by the increase in dioxygen concentra-
tion, whereas sod1D strain growth was severely restricted
a in the presence of high dioxygen in either carbon source.
N N In air, sod1D yeast grows to a lower saturation density
than wild-type yeast. Addition of 5 mM MnCl2 effected
Mn
a modest improvement in growth (20%) for the sod1D
O Cl O strain and a similarly sized decrease in growth for the
wild-type strain, such that their final densities were the
same (Fig. 1a). The effect of Mn2+ on sod1D yeast
R R
grown in 100% O2 was more striking. The addition of 5
Compound R group mM MnCl2 to SDC medium afforded full protection
EUK-8 H against the increase in O2 assault that is intolerable to
EUK-134 OCH3 the mutant in unsupplemented medium (Fig. 1b). Again,
EUK-189 OCH2CH3 5 mM Mn2+ caused a small decrease in growth in the
wild-type strain. Both wild-type and sod1D yeast grew to
b a lower 24-h optical density when glycerol was given as
the carbon source. When 5 mM MnCl2 was added to
N N
SGC medium, there was no significant effect on wild-
Cl Cl
type yeast, while sod1D yeast was again rescued to wild-
N
N
Mn
N
Mn
N
type levels (Fig. 1c).
N Cl N N Cl N

Temperature sensitivity
M40403 M40404
Wild-type yeast grew to a 30% lower density when
Structure 1 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 Fig. 2 Added Mn2+ affected growth of wild-type and sod1D yeast
more independent samples grown on the same day, with error bars incubated at elevated temperatures and extended survival of sod1D
indicating the standard deviation between samples. The experiment in the stationary phase. a The indicated strains were grown in SDC
was repeated at least three times and the data are representative of medium in the absence (white bars) or presence (black bars) of 5
these trials 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
(data not shown). By contrast, sod1D yeast exhibited a indicating the standard deviation between samples. The experiment
was repeated at least three times and the data are representative of
much more dramatic decrease in density after growth at these trials. b Wild-type EG103 and isogenic sod1D (EG118) strains
37C, reaching an OD600 only 4% of that observed after were incubated in SDC in the absence or presence of 5 mM MnCl2
the normal 30C incubation (data not shown). for 72 h. Cells were viability-tested by spotting 5·104, 5·103, 5·102,
Ionic manganese (5 mM MnCl2) was added to the and 5·101 cells onto YPD plates (1% yeast extract, 2% peptone,
2% dextrose, and 2% agar) and incubated at 30C in a low
cultures to test for rescue of this phenotype. In the case dioxygen environment. A characteristic result is shown. The
of wild-type yeast, the additional Mn2+ further re- experiment was repeated at least three times using three indepen-
duced the growth at 37C (Fig. 2a). However, the dent colonies and the same trend was observed
918

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

80
morphology (Fig. 3c). When sod1D yeast was grown in type B
the presence of 5 mM Mn2+, the fraction of sod1D cells 60
exhibiting type B morphology decreased to 36% (Fig. 40
3c). These values are similar to those observed for the
20
wild-type control and suggest that the addition of
exogenous manganese ions also rescues this phenotype. 0
In concert with previous findings suggesting that high wild type sod1∆ sod1∆+Mn
levels of manganese are somewhat detrimental to wild- Fig. 3 Growth in the presence of added Mn2+ results in sod1D
type yeast, 5 mM manganese added to the medium re- yeast with Leu1p activity, Aco1p activity, and vacuole morphology
sulted in a slight increase in vacuolar fragmentation in more similar to those of wild-type cells. a, b The indicated strains
wild-type yeast (data not shown). 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
EPR-detectable (free) iron and total iron levels after growth with or without 5 mM MnCl2 followed by growth
with 50 lM FM-4-64 as described in ‘‘Materials and methods.’’
The amount of ferric iron detectable by EPR at g=4.3 Type A vacuolar morphology refers to cells with one to two
vacuoles, which is considered normal, whereas a pattern with three
(FeIII
g=4.3) is roughly 5 times greater in EG118 yeast or more smaller vacuoles has been termed type B [44]. The average
(lacking CuZnSOD) than in its wild-type parent strain, of at least 400 cells from six colonies (three separate fields per
EG103 [14]. We measured the amount of FeIII
g=4.3 in these colony) is shown
919

a in the presence and absence of 5 mM MnCl2. While


100 the growth of wild-type yeast after 24 h was unaffected by

EPR-Detectable Iron (µM)


the presence of 2 mM ZnCl2 in the growth medium, the
80 sod1D yeast mutant exhibited a dramatic growth defect
60 [15]. The addition of 5 mM MnCl2 to growth medium
supplemented with 2 mM ZnCl2 had a small growth
40 inhibitory effect on wild-type yeast and a dramatic stim-
20 ulatory effect on sod1D yeast (90% growth enhancement).
The extra Mn2+ resulted in complete rescue of the zinc
0 sensitivity of sod1D yeast, as the mutant yeast reached the
wild type sod1∆ same OD600 as the wild-type yeast (Fig. 5).

b 300
Total Iron (µM)

Accumulation of zinc
200
We considered it possible that Mn2+ rescue results from
competition for binding and transport, resulting in a
100 reduction of the amount of zinc entering the cell. To
address this question, the zinc content was analyzed in
0 wild-type and sod1D yeasts grown for 24 h in SDC with
wild type sod1∆ or without 5 mM MnCl2, in the presence or absence of 1
mM ZnCl2. In these experiments, 1 mM ZnCl2 was used
Fig. 4 Growth in the presence of exogenous Mn2+ resulted in because the sod1D cells, although growth-inhibited, still
decreased levels of electron paramagnetic resonance (EPR) grew enough at this zinc concentration to provide suf-
detectable iron in sod1D yeast while total iron levels remained
relatively unchanged. The indicated strains were seeded at OD600 of
ficient sample for metal analysis. Without added zinc,
0.05 in minimal medium in the absence (white bars) or presence sod1D yeast accumulated almost twice as much zinc as
(black bars) of 5 mM MnCl2. Iron was measured after a 72-h wild-type yeast. However, in the presence of exogenous
incubation period. a Low-temperature iron EPR was employed to manganese, the amount of zinc in the two strains was the
measure the levels of FeIII
g=4.3 accumulation. b Total iron levels were same (Fig. 6a). In SDC with 1 mM ZnCl2, sod1D yeast
measured using a inductively coupled plasma atomic emission
spectrometer (ICP-AES) and are reported as the concentration of amassed approximately 25% more zinc. Supplementa-
iron. The values represent the average of two or more independent tion of SDC with 5 mM MnCl2 in addition to 1 mM
samples, with error bars indicating the standard deviation between Zn2+ resulted in a decrease in zinc accumulation in both
samples strains, again restoring wild-type zinc levels in the sod1D
mutant (Fig. 6b).
accumulated on average about 10% more total iron, but
the difference was not statistically significant (Fig. 4b).
Effect of Zn2+ on manganese accumulation

Accumulation of Mn2+ To test whether zinc toxicity is related to an effect of zinc


on intracellular manganese levels, the total manganese
We determined the intracellular concentration of Mn2+
in wild-type and sod1D yeasts in order to determine
10
Growth (OD at 600 nm)

whether a specific intracellular level of this metal was


required for the observed rescue. We used an ICP-AES to SDC
8
measure the manganese content in cells after growth SDC+5mM Mn
6
under our standard conditions (SDC pH 4.0, shaking in
air at 30C for 24 h) with or without the addition of 5 4
mM MnCl2. When no extra manganese was added, the
2
sod1D yeast accumulated similar levels of manganese to
wild-type cells. When yeast cells were grown on minimal 0
- Zn + Zn - Zn + Zn
medium supplemented with 5 mM MnCl2, both wild-
type and sod1D yeasts acquired high levels of manganese wild type sod1D
(Fig. 7). 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
Hypersensitivity to zinc 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
Wei et al. [15] demonstrated that 2 mM Zn2+ inhibited is an average of six colonies grown on two different days, with error
the growth of sod1D yeast. This experiment was repeated 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
treated samples.
5 SDC +5mM Mn
4
3
Growth in the presence of manganese-containing
2
SOD mimics
1
0
wild type sod1D A number of manganese-containing coordination com-
plexes, studied in cell culture and whole animals, were
b 200 shown to be effective in preventing damage attributed to
SDC +1mM Zn oxidative stress [20, 21, 46] (see ‘‘Introduction’’). On this
Total Zn (ppb/OD)

160 SDC +1mM Zn +5mM Mn basis, it has been assumed that these compounds are
120 acting as functional SOD mimics. To our knowledge, the
effect of manganese-containing SOD mimics has not
80
been studied previously in a yeast model system. We
40 therefore examined several manganese-containing SOD
0 mimics to determine their effectiveness in our yeast
wild type sod1D model system.
The Mn(III) salen derivatives from Eukarion are a
Fig. 6 Effect of manganese on zinc accumulation in wild-type and unique class of antioxidants that exhibit both SOD
sod1D yeasts. a Wild-type EG103 and isogenic sod1D yeasts were activity and catalase activity. We tested Euk-8, Euk-134,
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 and Euk-189, which differ in the nature of the substit-
mM ZnCl2 (gray bars) or in SDC with both 1 mM ZnCl2 and 5 mM uents on the salen moiety. They were reported to have
MnCl2 (white bars). In both experiments, cells were collected, the same level of SOD activity but differed in their levels
washed, and total zinc was measured with an ICP-AES and is of catalase activity and lipophilicity. The catalase
reported as the amount of zinc per optical density (approximately
107 cells). The values are the average of at least 12 samples,
activity of the prototypical Euk-8 (148 lM O2/min) was
harvested and analyzed on two different days. Error bars represent reported to be lower than that of Euk-134 or Euk-189
the standard deviation (243 and 180 lM O2/min, respectively), and Euk-189

content was measured in wild-type and sod1D strains a 0.4


after growth in SDC supplemented with 5 mM MnCl2, 1
Total Mn (ppb/OD)

mM ZnCl2, or both. Growth in SDC plus 1 mM ZnCl2 SDC


0.3
resulted in a reduction in manganese in wild-type and SDC +1mM Zn
sod1D yeasts by about 33% when compared with yeast 0.2
grown in SDC without added metals (Fig. 7a). This
influence of zinc on the total manganese content was 0.1
similar for wild-type and sod1D yeasts grown in SDC
0
plus 5 mM MnCl2; the strains accumulated about 20% wild type sod1D
less manganese when zinc was present in addition to the
excess manganese (Fig. 7b). b 20 SDC +5mM Mn
Total Mn (ppb/OD)

16 SDC +1mM Zn +5mM Mn


MnSOD activity 12
8
One possible explanation for our results could be that
excess manganese functions by activating the endoge- 4
nous MnSOD, which is still present in the mitochondrial 0
matrix of the sod1D strains. Two pieces of evidence ar- wild type sod1D
gue against this possibility. First, manganese rescue is
equally effective in an sod1Dsod2D double knockout Fig. 7 Effect of zinc on manganese accumulation in wild-type and
strain: MnCl2 fully rescues the lysine auxotrophy [17], 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
hypersensitivity to elevated temperature, and hypersen- bars). b The same strains were grown for 24 h in SDC with 5 mM
sitivity to ZnCl2 of the sod1Dsod2D double knockout MnCl2 (black bars) or in SDC with both 1 mM ZnCl2 and 5 mM
strain (data not shown). Second, we performed gel-based MnCl2 (white bars). In both experiments, cells were collected,
SOD activity assays on wild-type and sod1D strains (Fig. washed, and the total manganese was measured with an ICP-AES
and is reported as the amount of manganese per optical density
8). Although the sod1D strain appears to have somewhat (approximately 107 cells). The values are the average of at least 12
more MnSOD activity than the SOD1+ strain, the samples harvested and analyzed on two different days. Error bars
addition of manganese to the growth medium did not represent the standard deviation
921

Fig. 8 SOD activity. Native polyacrylamide gel electrophoresis of treatment to inhibit the CuZnSOD. The samples are wild-type
yeast extracts, showing endogenous SOD enzyme activity deter- yeast (1), wild-type yeast treated with 5 mM MnCl2 (2), sod1D yeast
mined by in-gel activity assay a after and b before cyanide (3), and sod1D yeast treated with 5 mM MnCl2 (4)

was found to be more lipophilic than either Euk-8 or iron: all of the known sod1D yeast growth phenotypes
Euk-134 [20]. were rescued fully, including the zinc sensitivity and the
The aerobic growth of wild-type and sod1D S. cere- iron-related defects such as reduced aconitase and Leu1p
visiae cultured in SDC pH 6.0 in the presence of each of activities and elevated levels of EPR-detectable iron.
the Eukarion compounds was recorded after 24 h. Wild- There are some systems in which the effect of elevated
type yeast cells were largely unaffected by treatment with levels of manganese may be attributed to an increase in
Euk-8, Euk-134, or Euk-189. The poor aerobic growth the activity of the mitochondrial MnSOD [48]. We have
of sod1D yeast was not improved upon treatment with found that this is not the case in yeast. First, we con-
any concentration of Euk-8, Euk-134, or Euk-189 tested, firmed the earlier observation that Mn2+ rescues the
in stark contrast to the rescue observed with ionic sod1Dsod2D double mutant, which lacks MnSOD [17].
Mn2+. Instead, relatively low concentrations of these Second, activity gels showed no increase in activity of
compounds prevented growth of these strains. We MnSOD in cells treated with high manganese (Fig. 8). If
interpret this effect to mean that the compounds are able anything, the activity of MnSOD was decreased by high
to enter the cells. manganese in both wild-type yeast and the sod1D mu-
In order to determine whether any beneficial effects of tant. S. cerevisiae normally requires only low levels (less
the Eukarion compounds might be observed under more than 3 lM) of manganese in the medium to sustain
stringent conditions, the 24-h aerobic growth of wild-
type and sod1D yeast in SDC pH 6.0 lacking lysine or a
methionine was monitored in the presence of up to 3 lM 10
Growth (OD at 600 nm)

Euk-134. Wild-type yeast was not affected by the pres- WT Euk-134


8 sod1∆ Euk-134
ence of Euk-134 in either drop-out medium. Very little
growth of sod1D S. cerevisiae in either SD-Lys or SD- 6 WT Euk-8
Met was observed, and the growth was not improved sod1∆ Euk-8
4
WT Euk-189
with the addition of Euk-134 (data not shown).
2 sod1∆ Euk-189
The Mn(III) cyclam derivative M40403 from Meta-
phor that we tested has been reported to be highly sta- 0
ble, with a high level of SOD activity and no catalase 1 10 100
Eukarion Concentration (µM)
activity [21, 47]. Aerobic growth of wild-type S. cerevi-
siae in the presence of M40403 remained unchanged b
from the untreated control as measured after 24 h (Fig.
Growth (OD at 600 nm)

10
9b). sod1D yeast cells cultured with M40403 were not 8
SDC
rescued by low levels of this mimic (0.05 and 5 lM, data active (M40403)
not shown), and, in fact, their growth was inhibited by 6 inactive (M40404)
about 90% with 25 lM M40403 (Fig. 9b). The struc- 4
turally related SOD-inactive control compound M40404
2
had no effect on wild-type yeast or mutant yeast at the
same concentrations. The observation of growth inhi- 0
bition for sod1D yeast cultured with M40403 is strong wild type sod1∆
evidence that this manganese complex enters yeast cells;
Fig. 9 The presence of Eukarion or Metaphore SOD mimics does
on the basis of their structural similarities, it seems likely not improve growth of sod1D Saccharomyces cerevisiae. Total
that M40404 enters yeast cells as well. 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),
Discussion 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
Our studies demonstrate that the beneficial effects of the untreated control (white bars). The values represent the average
manganese supplementation on sod1D yeast are much of six or more independent colonies grown on two or more days,
more dramatic and complete than those of copper or with error bars indicating the standard deviation
922

growth. It is interesting to note that in our experiments scavenging ability of Mn2+ present in the compartment.
the concentration of MnCl2 that provided the optimal We find that manganese does accumulate to high levels in
rescue of the sod1D yeast, i.e., 5 mM, was the same mitochondria of cells grown in high manganese (data not
concentration at which a small but significant negative shown), lending support to this hypothesis.
effect is first observed on control wild-type cells. Below The deleterious effect of the two classes of manga-
that concentration, the addition of MnCl2 to wild-type nese-containing SOD mimics, the manganese salen
cells had no effect on growth, and extremely high levels derivatives, Euk-8, Euk-134, and Euk-189, and the
of manganese (8-10 mM) were toxic. One possible manganese cyclam complex, M40403, on the growth of
explanation is that 5 mM is the lowest concentration of the sod1D yeast was quite surprising to us. In the case of
Mn2+ in the medium that provides a rate of influx of the manganese cyclam derivatives, we have the advan-
Mn2+ that is not totally counteracted by the homeo- tage of being able to compare two very similar coordi-
static mechanisms in place to handle toxic levels of nation complexes of manganese, one of which has high
exogenous metals. SOD activity and one of which does not. The SOD-
The rescue by manganese of the zinc sensitivity of the active compound, M40403, inhibited the growth of the
sod1D yeast was particularly striking to us, since it indi- sod1D yeast, while the SOD-inactive compound,
cates that the absence of the SOD1 polypeptide does not M40404, had no effect. The fact that M40403 inhibited
result in zinc sensitivity so long as the cell can otherwise growth is evidence that this compound does enter yeast
totally defend itself against superoxide. Mn2+ does not cells; the structural similarity of M40403 and M40404
appear to be competing with Zn2+ for import because make it likely that the latter compound enters yeast cells
intracellular zinc levels are not lowered when cells are as well. These results suggest that the growth inhibitory
grown in 5 mM MnCl2 (Fig. 7b). Thus, although the effect of the SOD-active manganese-containing com-
possibility remains that manganese displaces zinc from pounds on the sod1D yeast may be related to their SOD
some important site that mediates zinc toxicity without activity. It is possible that alterations in levels of ROS
changing cellular zinc levels, the present data appear to may lead to the disruption of cell signaling, resulting in
support the hypothesis that Mn2+ rescue of sod1D yeast poor growth, as the transcription of many genes is in-
is due primarily to its antioxidant action in vivo. In our duced by ROS [50, 51], but further work is required to
previous study [15], zinc resistance was not restored by evaluate this intriguing hypothesis. What is clear now
cytosolic expression of MnSOD from Bacillus stearo- from this study is that these two classes of manganese-
thermophilus; this bacterial enzyme did however rescue containing SOD mimetic compounds do not provide any
paraquat sensitivity [15]. This result seems contradictory benefit to the sod1D yeast, while the presence of ionic
to our current conclusion that Mn2+ rescue of sod1D Mn2+ provides full rescue.
yeast is due to its ability to scavenge superoxide. One The antioxidant role of ionic Mn2+ in vivo is thought
possible explanation is that Mn2+ may be capable of to be due to its superoxide scavenging ability, and, as
protecting in a subcellular compartment not accessible to discussed in the ‘‘Introduction’’, a number of prokary-
the bacterial MnSOD, a compartment that might be otic species are thought to use Mn2+ for exactly this
particularly sensitive to the adverse effects of high zinc. A purpose [1]. It now appears that this phenomenon is not
likely candidate is the IMS, for the following reasons. restricted to prokaryotes and that the simple eukaryote
Zinc and manganese can easily access this space via pores S. cerevisiae can use manganese in this way as well.
in the outer membrane that permit the passage of small Because Mn2+ rescues all the phenotypes tested, we are
water-soluble molecules, but proteins (i.e., the bacterial inclined to believe that it works by chemically removing
MnSOD) and other large molecules that are not sub- superoxide. Future studies will aim to get a better
strates for specific transport across the membrane are understanding of the subcellular localization and ligand
excluded. Superoxide is produced in the IMS, CuZnSOD environment as well as the inorganic chemistry of
is ordinarily present, and CuZnSOD in this area was manganese that underlies its remarkable antioxidant
reported to be important for long-term survival of yeasts properties when it is present at high concentrations in
[42]. High zinc is detrimental to many mitochondrial cells lacking SOD enzymes.
functions [49]. If this compartment is especially sensitive
to high levels of superoxide, then in the absence of active Acknowledgements This work was supported by grant DK46828 to
CuZnSOD it could be protected either by correct zinc J.S.V. We also gratefully acknowledge the support of the National
handling, insured by the zinc-binding function of inactive Science Foundation Predoctoral Fellowship (to R.J.S.), the NIH
Chemistry-Biology Interface Predoctoral Training grant (to R.J.S.
SOD1p (in yeast lacking the CCS1 (LYS7) gene,1ccs1D, and M.A.W.), and the University of California Toxic Substances
or sod1D yeast expressing H46C-SOD, an SOD-inactive, Research and Teaching Program, Lead Campus Program in Toxic
zinc-binding mutant of SOD1), or by the superoxide Mechanisms (to M.A.W.)

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