Features of Rhodobacter sphaeroides ChrR required for stimuli to promote the dissociation of σ(E)/ChrR complexes

• Published in: Journal of molecular biology 2011-04, Vol.407 (4), p.477
• Main Authors: Greenwell, Roger, Nam, Tae-Wook, Donohue, Timothy J
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Coenzymes - metabolism; Gene Expression Regulation; Models, Molecular; Mutagenesis, Site-Directed; Mutant Proteins - genetics; Mutant Proteins - metabolism; Protein Binding; Protein Structure, Quaternary; Rhodobacter sphaeroides - genetics; Rhodobacter sphaeroides - metabolism; Sigma Factor - metabolism; Singlet Oxygen - metabolism; Transcription Factors - genetics; Transcription Factors - metabolism; Transcription, Genetic; Zinc - metabolism
• ISSN: 1089-8638
• DOI: 10.1016/j.jmb.2011.01.055

In the photosynthetic bacterium Rhodobacter sphaeroides, a transcriptional response to the reactive oxygen species singlet oxygen ((1)O(2)) is mediated by ChrR, a zinc metalloprotein that binds to and inhibits the activity of the alternative σ factor σ(E). We provide evidence that (1)O(2) promotes the dissociation of σ(E) from ChrR to activate transcription in vivo. To identify what is required for (1)O(2) to promote the dissociation of σ(E)/ChrR complexes, we analyzed the in vivo properties of variant ChrR proteins with amino acid changes in conserved residues of the C-terminal cupin-like domain (ChrR-CLD). We found that (1)O(2) was unable to promote the detectable dissociation of σ(E)/ChrR complexes when the ChrR-CLD zinc ligands (His141, His143, Glu147, and His177) were substituted with alanine, even though individual substitutions caused a 2-fold to 10-fold decrease in zinc affinity for this domain relative to that for wild-type ChrR (K(d)∼4.6×10(-)(10) M). We conclude that the side chains of these invariant residues play a crucial role in the response to (1)O(2). Additionally, we found that cells containing variant ChrR proteins with single amino acid substitutions at Cys187 or Cys189 exhibited σ(E) activity similar to those containing wild-type ChrR when exposed to (1)O(2), suggesting that these thiol side chains are not required for (1)O(2) to induce σ(E) activity in vivo. Finally, we found that the same aspects of R. sphaeroides ChrR needed for a response to (1)O(2) are required for the dissociation of σ(E)/ChrR complexes in the presence of the organic hydroperoxide t-butyl hydroperoxide.