6533b861fe1ef96bd12c568d

RESEARCH PRODUCT

Staphylococcal NreB: an O2-sensing histidine protein kinase with an O2-labile iron-sulphur cluster of the FNR type

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Summary The nreABC ( n itrogen re gulation) operon encodes a new staphylococcal two-component regulatory sys- tem that controls dissimilatory nitrate/nitrite reduc- tion in response to oxygen. Unlike other two- component sensors NreB is a cytosolic protein with four N-terminal cysteine residues. It was shown that both the NreB-cysteine cluster and Fe ions are required for function. Isolated NreB was converted to the active form by incubation with cysteine desul- phurase, ferrous ions and cysteine. This activation is typical for FeS-containing proteins and was reversed by oxygen. During reconstitution an absorption band at 420 nm and a yellow-brownish colour (typical for an FNR-type iron-sulphur cluster formation) devel- oped. After alkylation of thiol groups in NreB and in the cysteine mutant NreB(C62S) almost no iron- sulphur cluster was incorporated; both findings corroborated the importance of the cysteine resi- dues. Comparison of the kinase activity of (i) the reconstituted (ii) the unreconstituted, and (iii) the unreconstituted and deferrated NreB-His indicated that NreB kinase activity depended on iron availabil- ity and was greatly enhanced by reconstitution. NreB is the first direct oxygen-sensing protein described in staphylococci so far. Reconstituted NreB contains 4- 8 acid-labile Fe and sulphide ions per NreB which is in agreement with the presence of 1-2 iron-sulphur (4Fe-4S)