The Response Regulator RegA Is a Copper Binding Protein That Covalently Dimerizes When Exposed to Oxygen
2022
Nijia Ke | Carl E. Bauer
In Rhodobacter capsulatus, the histidine kinase RegB is believed to phosphorylate its cognate transcriptional factor RegA only under anaerobic conditions. However, transcriptome evidence indicates that RegA regulates 47 genes involved in energy storage, energy production, signaling and transcription, under aerobic conditions. In this study, we provide evidence that RegA is a copper binding protein and that copper promotes the dimerization of RegA under aerobic conditions. Inductively coupled plasma mass spectrometry (ICP-MS) analysis indicates that RegA binds Cu<sup>1+</sup> and Cu<sup>2+</sup> in a 1:1 and 2:1 ratio, respectively. Through LC-MS/MS, ESI-MS and non-reducing SDS-PAGE gels, we show that Cu<sup>2+</sup> stimulates disulfide bond formation in RegA at Cys156 in the presence of oxygen. Finally, we used DNase I footprint analysis to demonstrate that Cu<sup>2+</sup>-mediated covalent dimerized RegA is capable of binding to the ccoN promoter, which drives the expression of cytochrome cbb<sub>3</sub> oxidase subunits. This study provides a new model of aerobic regulation of gene expression by RegA involving the formation of an intermolecular disulfide bond.
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