Abstract
The transcription factor FNR (fumarate nitrate reduction) requires
the presence of an iron-sulfur (Fe-S) cluster for its function as
a global transcription regulator in Escherichia coli when oxygen
becomes scarce. To define the oxidation state and type of Fe-S cluster
present in the active form of FNR, we have studied anaerobically
purified FNR with Mössbauer spectroscopy. Our data showed that
this form of FNR contained a 4Fe-4S2+ cluster (delta = 0.45 mm/s;
DeltaEQ = 1.22 mm/s) and that the 4Fe-4S2+ cluster was rapidly
destroyed on exposure of FNR to air. Under these conditions, the
yellow-green active form of FNR turned deep red; analysis of sulfide
indicated that 70% of the labile sulfide was still present, suggesting
that the Fe-S cluster had been converted into a different form.
Little 3Fe-4S cluster was, however, detected by EPR. According
to Mössbauer spectroscopy, the 4Fe-4S2+ cluster was converted
in about 60% yield to a 2Fe-2S2+ cluster (delta = 0.28 mm/s; DeltaEQ
= 0.58 mm/s) following 17 min of exposure to air. The 2Fe-2S2+
cluster form of FNR was much more stable to oxygen, but was unable
to sustain biological activity (e.g., DNA binding). However, DNA
binding and the absorption spectrum characteristic of the 4Fe-4S2+
cluster could be largely restored from the 2Fe-2S2+ form when
Cys, Fe, DTT, and the NifS protein were added. It has yet to be
determined whether the form of FNR containing the 2Fe-2S2+ cluster
has any biological significance, e.g., as an in vivo intermediate
that is more rapidly converted to the active form than the apoprotein.
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