Fate of Oxo-Histidine Containing Proteins

National Heart, Lung, And Blood Institute

Investigators

Abstract

We elected to utilize the bacterial transcription factor PerR as the substrate for putative oxo-histidine reductases. In Bacillus subtilis, PerR is known to undergo oxidative modification when cells are exposed to oxidative stress. The oxidation serves as a switch for the transcription factor. Helmann and colleagues established that the oxidative modification is conversion of one or two histidine residues to oxohistidine. We have now cloned and purified PerR and made its oxohistidine form. These standards allowed development of an isoelectric focusing technique to separate the native and oxidized forms from cell extracts. Upon application of hydrogen peroxide to cells, the intracellular PerR was immediately converted to the oxidized form. Following removal of the peroxide, the total PerR level was unchanged, but the oxidized form was removed within 15-30 min. We are now determining whether this clearance is a consequence of degradation of the oxidized form and synthesis of new native PerR or whether the oxohistidine was reduced back to histidine.