Affiliation:
1. Institute for Microbiology and Wine Research, University of Mainz, Becherweg 15, 55099 Mainz, Germany
2. Roche Diagnostics GmbH, Rare Reagents R&D/Chemistry, 82377 Penzberg, Germany
Abstract
ABSTRACT
The sensor kinase NreB from
Staphylococcus carnosus
contains an O
2
-sensitive [4Fe-4S]
2+
cluster which is converted by O
2
to a [2Fe-2S]
2+
cluster, followed by complete degradation and formation of Fe-S-less apo-NreB. 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 labeling procedure, accessible Cys residues in the native protein were first labeled by iodoacetate. In the second step, Cys residues not labeled in the first step were alkylated with the fluorescent monobromobimane (mBBr) after denaturing of the protein. In purified (aerobic) apoNreB, most (96%) of the Cys residues were alkylated in the first step, but in anaerobic (Fe-S-containing) NreB only a small portion (23%) were alkylated. In anaerobic bacteria, a very small portion of the Cys residues of NreB (9%) were accessible to alkylation in the native state, whereas most (89%) of the Cys residues from aerobic bacteria were accessible. The change in accessibility allowed determination of the half-time (6 min) for the conversion of NreB·[4Fe-4S]
2+
to apoNreB after the 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 labeling experiments.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
14 articles.
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