From the Department of Biochemistry, Duke University Medical
Center, Durham, North Carolina 27710
Enrichment of the growth medium with iron
partially relieves the phenotypic deficits imposed on Escherichia
coli by lack of both manganese and iron superoxide dismutases.
Thus iron supplementation increased the aerobic growth rate, decreased
the leakage of sulfite, and diminished sensitivity toward paraquat.
Iron supplementation increased the activities of several
[4Fe-4S]-containing dehydratases, and this was seen even in the
presence of 50 µg/ml of rifampicin, an amount which completely
inhibited growth. Assessing the O
2 scavenging activity by
means of lucigenin luminescence indicated that the iron-enriched
sodAsodB cells had gained some means of eliminating O
2, which was not detectable as superoxide
dismutase activity in cell extracts. It is noteworthy that
iron-enriched cells were not more sensitive toward the lethality of
H2O2 despite having the usual amount of
catalase activity. This indicates that iron taken into the cells from
the medium is not available for Fenton chemistry, but is available for
reconstitution of iron-sulfur clusters.
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INTRODUCTION |
Among the targets susceptible to direct oxidation by O
2
are those dehydratases that contain [4Fe-4S] clusters. These enzymes, which include dihydroxy acid dehydratase (1-3), aconitase (4, 5),
6-phosphogluconate dehydratase (6-7), and fumarases A and B (8, 9),
react with O
2 with rate constants of ~107
M
1 s
1. Univalent oxidation of
the [4Fe-4S] clusters by O
2 leads to loss of iron, leaving
[3Fe-4S] clusters and an inactive form of the enzymes. The enzymes
can subsequently be reactivated by reductive reconstitution (10, 11).
Both inactivation and reactivation are ongoing processes in aerobic
cells and their balance determines the fractional level of activity of
these enzymes (12, 13).
One of the functions of superoxide dismutases is to protect these
dehydratases against this inactivation by O
2. However, there
are other means by which O
2 can cause damage, both directly and indirectly (14, 15). This leads to the possibility that consumption
of O
2 by the reversible inactivation of the dehydratases may
serve to prevent these other kinds of damage. In that case, increasing
the rates of reactivation of the dehydratases by growing the cells
in iron-enriched medium may partially reverse the phenotypic deficits
of sodA sodB Escherichia coli. The results reported herein are consistent with that outcome.
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MATERIALS AND METHODS |
Bound sulfite was liberated with alkaline cyanide as described
by Kunert (16) and used recently (17). The liberated sulfite was
assayed colorimetrically (17, 18). The strains of E. coli used in these experiments were: AB1157, parental; JI132, sodA sodB (19); and pLK1646p GS57, fumA fumC, and
overproducing fumarase A by virtue of a multicopy plasmid bearing the
fumA gene (20). Unless otherwise indicated starter cultures
were grown overnight in LB1
and were then used to inoculate M9CA medium at a dilution of 1:200. LB
and M9CA were prepared as described by Maniatis et al. (21).
Growth in defined medium was achieved by washing cells taken from the
overnight cultures 3 times in M9 followed by 200-fold dilution into M9
supplemented with 100 mg/liter of the 20 amino acids, except methionine
and cysteine, commonly found in proteins (referred to as 18AA medium).
In each case the defined medium also contained 0.2% glucose plus 3 mg/liter each of pantothenic acid and thiamin. Minimal medium contained
the vitamins mentioned above plus 100 mg/liter of Thr, Leu, His, Pro,
and Arg, and 0.2% glucose. The inocula for growth in minimal medium
were taken from overnight anaerobic cultures in this medium. Iron was
added as water solutions of FeSO4 or FeCl2
sterilized by filtration. Anaerobiosis was achieved in a Coy chamber.
Aerobic growth was at 200 rpm. All growth was at 37 °C and was
followed turbidimetrically at 600 nm. Iron was determined
colorimetrically with
,
'-dipyridyl applied to cell extracts (22).
Fumarase activity was assayed according to Hill and Bradshaw (23) and
catalase activity, as described by Beers and Sizer (24). Aconitase (4,
25), dihydroxy acid dehydratase (1), and lucigenin luminescence (26)
were assayed as described previously.
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RESULTS |
Iron Improves the Aerobic Growth of sodA sodB E. coli--
Adding
Fe(II) to the M9CA medium hastened the aerobic growth of the sodA
sodB strain. Fe(II), 0.05-0.50 mM, caused a
dose-dependent increase in growth. Thus the generation
time, which was 84 min in the absence of added Fe(II), was shortened to
72 min by 0.1 mM Fe(II) and to 56 min by 0.5 mM
Fe(II). The doubling time of the parental strain was 44 min without
iron supplementation. The sodA sodB strain grew very slowly
in medium lacking the aromatic, branched chain, and
sulfurcontaining amino acids, and 0.5 mM Fe(II) exerted
a very substantial stimulatory effect. Thus the doubling time, which
was ~630 min without iron, was shortened to 112 min by 0.5 mM Fe(II). Supplementation with 0.5 mM Co(II)
or Cu(II) was without effect, whereas 0.5 mM Mn(II) was
beneficial but not as much as 0.5 mM Fe(II) (data not
shown). Mn(II) has been reported to act as a functional replacement for
superoxide dismutase in Lactobacillus plantarum (27) and in
yeast (28), and mutations in yeast causing accumulation of Mn(II)
complemented a lack of the CuZn-superoxide dismutase (29). This effect
of Mn(II) can be attributed to the ability of Mn(II) and complexes
thereof to catalyze the dismutation and/or the reduction of O
2
(30, 31). Mn(II) does not participate in Fenton chemistry (32, 33), whereas Fe(II) does. Hence a protective effect of Mn(II) is not surprising, but a protective effect by Fe(II) demands explanation.
Iron Diminishes the Leakage of Sulfite--
The sodA sodB E. coli exhibits an oxygen-dependent requirement for
sulfurcontaining amino acids (34) and this has been attributed to
the leakage of sulfite from the cells (17, 35). Fig.
1 shows that 0.5 mM Fe(II) in
the 18AA medium diminished the accumulation of sulfite by the
sodA sodB strain (compare bars 1 and
2). Incubation of the medium conditioned by the growth of
the sodA sodB cells with 0.5 mM Fe(II) for
24 h did not significantly diminish recovery of sulfite, probably
because the sulfite was present as a carbonyl-bisulfite adduct (17) and
thus was not subject to rapid metal-catalyzed autoxidation. In any case
the data in Fig. 1 have been corrected for the effect of Fe(II) on the
recovery of sulfite. Hence the effect of Fe(II) was to decrease leakage
of sulfite and not to diminish recovery of sulfite from the medium. As
expected there was little leakage of sulfite from the parental strain
growing aerobically (bar 4) or from the sodA sodB
growing anaerobically (bar 3).

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Fig. 1.
Leakage of sulfite. Cultures in the 18AA
medium were incubated for 24 h, and the conditioned medium was
then assayed for cryptic sulfite (16). Bar 1, sodA
sodB aerobic; bar 2, sodA sodB + 0.5 mM Fe(II), aerobic; bar 3, sodA sodB,
anaerobic; bar 4, parental strain, aerobic.
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Fe(II) Supplementation Protects against Paraquat--
The
sodA sodB strain is ~100 times more sensitive to paraquat
than the superoxide dismutase-replete parental strain (34). Fig.
2 shows the strong growth inhibition
imposed by 2 and 4 µM paraquat on the sodA
sodB strain (compare lines 4 and 5 with
3). Supplementation with Fe(II) eliminated the effect of
paraquat and even allowed faster growth than that seen without paraquat (compare lines 1 and 2 with 3). These
low micromolar levels of paraquat were without effect on the growth of
the parental strain (data not shown).

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Fig. 2.
Iron protects against paraquat.
Overnight cultures of sodA sodB in LB were diluted 200-fold
into M9CA with the following additions: line 1, 2.0 µM paraquat + 0.5 mM Fe(II); line
2, 4.0 µM paraquat + 0.5 mM Fe(II);
line 3, none; line 4, 2 µM
paraquat; line 5, 4.0 µM paraquat.
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Growth in iron-supplemented medium did result in greater cell content
of iron and the sodA sodB strain accumulated more iron than
did the parental strain. Thus both strains contained 2 µmol of
iron/mg of protein when grown without added iron, but when grown in the
presence of 0.5 mM Fe(II) the sodA sodB
contained 16 and the parental strain 5.5 µmol of iron/mg of protein.
The sodA sodB strain was more susceptible to the lethality
of H2O2 than the parental strain as has been
reported (34). However, growth in the presence of 0.5 mM
Fe(II) did not increase the sensitivity of the sodA sodB
strain to H2O2. Thus 30 min of exposure to 2.0 mM H2O2 at 37 °C caused ~75%
loss of viability of washed cells suspended in M9 salts at
A600 nm = 0.5 whether or not they had been
grown in Fe(II)-supplemented medium.
Luminescence--
Although the luminescence elicited from
lucigenin is not a reliable measure of the concentration of O
2
because it can mediate production of O
2, it can be used to
detect the scavenging of O
2 by superoxide dismutase or by
other means (36, 37). Fig. 3 demonstrates
that the sodA sodB cells grown in iron-supplemented medium
luminesce less than when grown without such supplementation. The
parental strain luminesced much less than the sodA sodB
strain as previously noted (26). It thus appears that the sodA
sodB cells gained some mechanism of scavenging O
2 when
grown in iron-enriched medium. Washing the cells prior to addition of
lucigenin increased luminescence, but the difference between the
iron-rich and normal sodA sodB cells persisted, as did the
striking difference between the sodA sodB and parental
cells.

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Fig. 3.
Lucigenin luminescence. Mid-log cultures
in M9CA ± 0.5 mM Fe(II) were collected by
centrifugation and then half were washed with M9 medium and half were
not. The cells were suspended to A600 nm in 100 mM potassium phosphate, 0.2% glucose, and 0.2 mM lucigenin, and luminescence was recorded. Bars
1-3 (washed cells), 1 = parental strain;
2 = sodA sodB, iron-supplemented;
3 = sodA sodB, unsupplemented; bars
4-6 (unwashed cells), 4 = parental;
5 = sodA sodB, iron supplemented;
6 = sodA sodB, not supplemented.
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[4Fe-4S]-containing Dehydratases--
Growth of the
sodA sodB strain in iron-supplemented medium increased cell
content of several [4Fe-4S]- containing dehydratase activities. Thus
Fig. 4 demonstrates this for aconitase
and for dihydroxy acid dehydratase, whereas Fig.
5 makes this point for fumarases A + B in
the sodA sodB strain and for fumarase A in a FumA
overproducing strain. The data in Figs. 4 and 5 were obtained with
cells grown in M9CA medium with and without iron supplementation because that is the medium in which iron supplementation speeded the
growth of the sodA sodB strain. It should be noted that the sodA sodB strain grew better in LB than in M9CA, contained
~10 times more aconitase, and did not show a further growth
enhancement with iron supplementation.

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Fig. 4.
Aconitase and dihydroxy acid
dehydratase. The sodA sodB strain was grown in
M9CA ± 0.5 mM Fe(II) to
A600 nm = 1.0. The cells were harvested,
extracted, and assayed as described under "Materials and Methods."
In the case of cultures intended for aconitase assays, the effect of 50 µg/ml of rifampicin was tested by adding this antibiotic 15 min
before adding the iron. The rifampicin was added as a solution in
ethanol such that the final concentration of ethanol was less than 1%.
Bar 1, without iron; bar 2, with 50 µg/ml
rifampicin; bar 3, with 0.5 mM Fe(II); bar
4, with 0.5 mM Fe(II) and 50 µg/ml rifampicin.
Dihydroxy acid dehydratase: bar 1, without iron; bar
2, with 0.5 mM Fe(II).
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Fig. 5.
Fumarases. The sodA sodB and
fumarase-overproducing strain were grown to
A600 nm = 1.0 in M9CA ± 0.5 mM Fe(II). The washed cells were extracted and assayed for
fumarases A + B as described under "Materials and Methods."
Bars 1 = without Fe(II), and bars 2 = with Fe(II).
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Whether iron supplementation increased the activities of the
[4Fe-4S]-containing dehydratases because of increased reactivation of
the oxidatively inactivated enzymes or because it led to more de
novo synthesis was explored with an inhibitor of transcription i.e. rifampicin. Iron, at 0.5 mM, increased
the activity of aconitase in the sodA sodB strain by
3-fold. Rifampicin at 50 µg/ml, which completely inhibited growth,
did not diminish the stimulatory effect of iron (Fig. 4). It
follows that the effect of iron was due to reactivation rather than to
de novo synthesis. These experiments were performed by
adding rifampicin to mid-log cultures in M9CA and then, after 15 min,
adding Fe(II) to 0.5 mM to half of the cultures. After
further incubation for 90 min, during which
A600 nm did not increase, cells were washed
three times with 50 mM Tris at pH 7.6 and extracted
and assayed for aconitase as described under "Materials and
Methods."
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DISCUSSION |
Because of its role in Fenton chemistry, one might anticipate that
growing sodA sodB E. coli in iron-supplemented medium would make them more susceptible to oxidative stress. The data presented above show the opposite effect. Thus ironsupplemented cells grew faster in aerobic M9CA medium, leaked less sulfite, and were less susceptible to the growth inhibiting effect of paraquat than
unsupplemented cells. The observation that less lucigenin luminescence
was seen with the iron-supplemented cells indicates that they had an
enhanced O
2-scavenging activity. However, no superoxide
dismutase activity could be detected in extracts of the
iron-supplemented cells.
One possibility for the seat of this activity within the cells would be
an increased rate of reductive reconstitution of oxidatively inactivated [4Fe-4S]-containing dehydratases. The cycle of oxidative inactivation by O
2, followed by reductive reactivation of such clusters, could act as a sink for O
2. In keeping with this
view, the iron-grown sodA sodB cells contained
more aconitase, dihydroxy acid dehydratase, and fumarases A and B
activities than cells grown without iron enrichment. Rifampicin, an
inhibitor of transcription, did not eliminate this effect of iron on
aconitase activity. It follows that the increase in aconitase, caused
by iron supplementation, was mainly due to increased reactivation
rather than de novo synthesis.
Increased reactivation would provide two benefits. One is to provide a
sink for O
2 that could spare other, nonreactivatable targets
of O
2 attack. A second benefit would be to maintain the activities of these essential dehydratases high enough to meet the
metabolic needs of the cells. Of course such reductive scavenging of
O
2 is inferior to that achieved by superoxide dismutase in that it produces one H2O2 per O
2
consumed and also consumes cellular reductants such NADPH. In contrast,
superoxide dismutase produces only 0.5 H2O2 per
O
2 consumed and without consumption of reductants.
The failure of iron supplementation to increase the
H2O2 sensitivity of E. coli is
surprising in view of the demonstrated role of iron released from
O
2-oxidized [4Fe-4S] clusters in exacerbating the lethality
of H2O2 (38). It appears that iron taken into the cells from the medium is not available to participate in Fenton chemistry, although competent to reconstitute [4Fe-4S] clusters. The
relatively trivial explanation that iron supplementation increased catalase activity to a degree that offset increased toxicity was examined and eliminated. Thus the catalase activity of the sodA sodB cells grown with 0.5 mM Fe(II) was equal to that
of the control cells (data not shown).
We are indebted to Dr. J. R. Guest for
the FumA-overproducing strain of E. coli.