JB Accepts, published online ahead of print on 23 October 2009

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J. Bacteriol. doi:10.1128/JB.01248-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Response of the oxygen sensor NreB to air in vivo: Fe-S containing and apoNreB in aerobically and anaerobically growing Staphylococcus carnosus

F. Reinhart, A. Huber, R. Thiele, and G. Unden^*

Institute for Microbiology and Wine Research, University of Mainz, 55099 Mainz, Becherweg 15, Germany; Roche Diagnostics GmbH, Rare Reagents R&D/Chemistry, 82377 Penzberg, Germany

^* To whom correspondence should be addressed. Email: unden{at}uni-mainz.de.

The sensor kinase NreB from Staphylococcus carnosus contains an O₂ sensitive [4Fe-4S]²⁺ cluster which is converted by O₂ to a [2Fe-2S]²⁺ cluster followed by complete degradation and formation of FeS-less apoNreB. NreB^.[2Fe-2S]²⁺ and apoNreB are devoid of kinase activity. NreB contains four Cys residues which ligate the Fe-S clusters. The accessibility of the Cys residues to alkylating agents was tested and used to differentiate Fe-S-containing and Fe-S-less NreB. In a two-step labelling procedure accessible Cys residues were first labelled in the native protein by iodoacetate. In a second step, Cys residues not labelled in the first step, were alkylated with the fluorescent monobromobimane (mBBr) after denaturing the protein. In purified (aerobic) apoNreB most (96 %) of the Cys residues were alkylated in the first step, in anaerobic (Fe-S containing) NreB only a small portion (23%). In anaerobic bacteria a very small portion of the Cys residues of NreB (9%) was accessible to alkylation in the native state, whereas most (89%) of the Cys residues from aerobic bacteria were accessible. The change in accessibility allowed determining the half-time (6 min) for the conversion of NreB^.[4Fe-4S]²⁺ to apoNreB after addition of air in vitro. Overall, in anaerobic bacteria most of the NreB exists as NreB^.[4Fe-4S]²⁺, whereas in aerobic bacteria the (Fe-S-less) apoNreB is predominant and represents the physiological form. The number of accessible Cys residues was also determined by iodoacetate alkylation followed by mass spectrometry of Cys containing peptides. The pattern of mass increases confirmed the results from the two-step labelling experiments.