Iron-sulfur cluster disassembly in the FNR protein of Escherichia coli by O-2: [4Fe-4S] to [2Fe-2S] conversion with loss of biological activity

被引:286
作者
Khoroshilova, N
Popescu, C
Munck, E
Beinert, H
Kiley, PJ
机构
[1] UNIV WISCONSIN, SCH MED, DEPT BIOMOL CHEM, MADISON, WI 53706 USA
[2] CARNEGIE MELLON UNIV, DEPT CHEM, PITTSBURGH, PA 15213 USA
[3] UNIV WISCONSIN, INST ENZYME RES, GRAD SCH, MADISON, WI 53705 USA
[4] UNIV WISCONSIN, COLL AGR & LIFE SCI, DEPT BIOCHEM, MADISON, WI 53705 USA
关键词
transcriptional control; anaerobic metabolism; labile sulfide; oxygen sensing; Mossbauer spectra;
D O I
10.1073/pnas.94.12.6087
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 Mossbauer spectroscopy, Our data showed that this form of FNR contained a [4Fe-4S](2+) cluster (delta = 0.45 mm/s; Delta E-Q = 1.22 mm/s) and that the [4Fe-4S](2+) 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 Mossbauer spectroscopy, the [4Fe-4S](2+) cluster was converted in about 60% yield to a [2Fe-2S](2+) cluster (delta = 0.28 mm/s; Delta E-Q = 0.58 mm/s) following 17 min of exposure to air, The [2Fe-2S](2+) 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-4S](2+) cluster could be largely restored from the [2Fe-2S](2+) form when Cys, Fe, DTT, and the NifS protein were added. It has yet to be determined whether the form of FNR containing the [ZFe-2S](2+) 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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页码:6087 / 6092
页数:6
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