Abstract
Iron-sulfur proteins are among the sensitive targets of the nitric oxide cytotoxicity. When Escherichia coli cells are exposed to nitric oxide, iron-sulfur clusters are modified forming protein-bound dinitrosyl iron complexes. Such modified protein dinitrosyl iron complexes are stable in vitro but are efficiently repaired in aerobically growing E. coli cells even without any new protein synthesis. Here we show that cysteine desulfurase encoded by the gene iscS of E. coli can directly convert the ferredoxin dinitrosyl iron complex to the ferredoxin [2Fe-2S] cluster in the presence of L-cysteine in vitro. A reassembly of the [2Fe-2S] cluster in the ferredoxin dinitrosyl iron complex does not require any addition of iron or other protein components. Furthermore, a complete removal of the dinitrosyl iron complex from ferredoxin prevents reassembly of the [2Fe-2S] cluster in the protein. The results suggest that cysteine desulfurase (IscS) together with L-cysteine can efficiently repair the nitric oxide-modified ferredoxin [2Fe-2S] cluster and that the iron center in the dinitrosyl iron complex may be recycled for the reassembly of iron-sulfur clusters in proteins.
Highlights
The protein dinitrosyl iron complexes are stable in vitro, they are efficiently repaired in aerobically growing E. coli cells even in the presence of the protein synthesis inhibitor chloramphenicol [10, 14]
The results suggest that cysteine desulfurase (IscS) together with L-cysteine can efficiently repair the nitric oxide-modified ferredoxin [2Fe-2S] cluster and that the iron center in the dinitrosyl iron complex may be recycled for the reassembly of iron-sulfur clusters in proteins
In a search for such reducing equivalents, we found that L-cysteine, but not N-acetyl-L-cysteine or reduced glutathione, can effectively decompose the protein dinitrosyl iron complexes in the cell extracts prepared from the E. coli cells treated with nitric oxide [10]
Summary
Preparation of IscS—The coding region of the E. coli IscS protein was amplified from wild-type E. coli genomic DNA by PCR using PCR ready-to-go beads (Amersham Biosciences). Two primers were designed to contain a NcoI restriction site in one primer and a HindIII site in the other. The sequences of the primers are: IscS-l, 5Ј-GCATTGAGCCATGGACGGAGTTTA-3Ј; and IscS-2, 5Ј-CCGATTAAAGCTTAGCCCATT-
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