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F. WAYNE OUTTEN, PhD
Djaman
O, Outten FW, Imlay JA. Repair of oxidized iron-sulfur
clusters in Escherichia coli. J Biol Chem. 2004 Oct
22;279(43):44590-9.
The [4Fe-4S]2+ clusters of dehydratases
are rapidly damaged by univalent oxidants, including
hydrogen peroxide, superoxide, and peroxynitrite. The loss
of an electron destabilizes the cluster, causing it to
release its catalytic iron atom and converting the cluster
initially to an inactive [3Fe-4S]1+ form. Continued exposure
to oxidants in vitro leads to further iron release.
Experiments have shown that these clusters are repaired in
vivo. We sought to determine whether repair is mediated by
either the Isc or Suf cluster-assembly systems that have
been identified in Escherichia coli. We found that all the
proteins encoded by the isc operon were critical for de novo
assembly, but most of these were unnecessary for cluster
repair. IscS, a cysteine desulfurase, appeared to be an
exception: although iscS mutants repaired damaged clusters,
they did so substantially more slowly than did wild-type
cells. Because sulfur mobilization should be required only
if clusters degrade beyond the [3Fe-4S]1+ state, we used
whole cell EPR to visualize the fate of oxidized enzymes in
vivo. Fumarase A was overproduced. Brief exposure of cells
to hydrogen peroxide resulted in the appearance of the
characteristic [3Fe-4S]1+ signal of the oxidized enzyme.
When hydrogen peroxide was then scavenged, the enzyme
activity reappeared within minutes, in concert with the
disappearance of the EPR signal. Thus it is unclear why IscS
is required for efficient repair. The iscS mutants grew
poorly, allowing the possibility that metabolic defects
indirectly slow the repair process. Our data did indicate
that damaged clusters decompose beyond the [3Fe-4S]1+ state
in vivo when stress is prolonged. Under the conditions of
our experiments, mutants that lacked other repair
candidates--Suf proteins, glutathione, and NADPH: ferredoxin
reductase--all repaired clusters at normal rates. We
conclude that the mechanism of cluster repair is distinct
from that of de novo assembly and that this is true because
mild oxidative stress does not degrade clusters in vivo to
the point of presenting an apoenzyme to the de novo
cluster-assembly systems.
Outten FW, Djaman O, Storz G. A suf
operon requirement for Fe-S cluster assembly during iron
starvation in Escherichia coli. Mol Microbiol. 2004
May;52(3):861-72.
The suf and isc operons of Escherichia
coli have been implicated in Fe-S cluster assembly. However,
it has been unclear why E. coli has two systems for Fe-S
cluster biosynthesis. We have examined the regulatory
characteristics and mutant phenotypes of both operons to
discern if the two operons have redundant functions or if
their cellular roles are divergent. Both operons are
similarly induced by hydrogen peroxide and the iron chelator
2,2'-dipyridyl, although by different mechanisms. Regulation
of the isc operon is mediated by IscR, whereas the suf
operon requires OxyR and IHF for the response to oxidative
stress and Fur for induction by iron starvation.
Simultaneous deletion of iscS and most suf genes is
synthetically lethal. However, although the suf and isc
operons have overlapping functions, they act as distinct
complexes because the SufS desulphurase alone cannot
substitute for the IscS enzyme. In addition, suf deletion
mutants are more sensitive to iron starvation than isc
mutants, and the activity of the Fe-S enzyme gluconate
dehydratase is diminished in the suf mutant during iron
starvation. These findings are consistent with the model
that the isc operon encodes the housekeeping Fe-S cluster
assembly system in E. coli, whereas the suf operon is
specifically adapted to synthesize Fe-S clusters when iron
or sulphur metabolism is disrupted by iron starvation or
oxidative stress.
Outten FW, Wood MJ, Munoz FM, Storz G.
The SufE protein and the SufBCD complex enhance SufS
cysteine desulfurase activity as part of a sulfur transfer
pathway for Fe-S cluster assembly in Escherichia coli. J
Biol Chem. 2003 Nov 14;278(46):45713-9.
The sufABCDSE operon of the Gram-negative
bacterium Escherichia coli is induced by oxidative stress
and iron deprivation. To examine the biochemical roles of
the Suf proteins, we purified all of the proteins and
assayed their effect on SufS cysteine desulfurase activity.
Here we report that the SufE protein can stimulate the
cysteine desulfurase activity of the SufS enzyme up to
8-fold and accepts sulfane sulfur from SufS. This sulfur
transfer process from SufS to SufE is sheltered from the
environment based on its resistance to added reductants and
on the analysis of available crystal structures of the
proteins. We also found that the SufB, SufC, and SufD
proteins associate in a stable complex and that, in the
presence of SufE, the SufBCD complex further stimulates SufS
activity up to 32-fold. Thus, the SufE protein and the
SufBCD complex act synergistically to modulate the cysteine
desulfurase activity of SufS. We propose that this sulfur
transfer mechanism may be important for limiting sulfide
release during oxidative stress conditions in vivo.
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