Activation and Complex Regulation of the Kef Potassium Efflux System During Protection of Bacteria Against Toxic Electrophiles

Abstract

Electrophiles react rapidly with DNA and proteins and are therefore toxic. Bacteria protect themselves from the immediate effects of electrophiles by lowering the cytosolic pH before the compounds are detoxified. This is achieved by the Kef systems, which mediate potassium efflux, in turn coupled to an influx of protons. We investigated the activation and complex regulation of this system. When electrophiles enter the cell, the most likely reaction partner is the nucleophile glutathione which is present at high concentration in the cytosol. The resulting glutathione adducts activate Kef while glutathione itself inhibits. The kinetics of activation was investigated in vivo using a potassium selective electrode. Activator and inhibitor bind to the same binding site on the regulatory KTN domain of Kef and cause differential structural changes, as shown by crystal structures of this domain [1]. We characterised the binding and these structural changes further using fluorescence and EPR spectroscopy in purified Kef proteins. A second binding site on the KTN domain binds nucleotides. Furthermore, Kef is regulated by a soluble protein which serves as activator [2]. We present data that this activator protein also has quinone oxidoreductase activity. The relationship between this enzymatic activity and its function as an activator of Kef was dissected using point mutations to disrupt enzyme activity but preserve structural integrity.[1] Rossild et al., Structure 17, 893 (2009).[2] Miller et al., J. Bac.182, 6536 (2000).