title: Conformational changes in the novel redox sensor protein HbpS studied by site-directed spin labeling and its turnover in dependence on the catalase-peroxidase CpeB.
abstract: OBJECTIVE
  To establish conditions to study the oligomeric assembly of heme-binding protein (HbpS) in solution by applying the tools of site-directed spin labeling combined with pulse electron paramagnetic resonance (SDSL EPR) spectroscopy, as well as to analyze redox stress-based conformational changes in HbpS subunits within the oligomer in solution. In vivo elucidation of molecular mechanisms that control the downregulation of the novel redox-system HbpS-SenS-SenR.
  
  
  RESULTS
  Using a set of specifically generated HbpS mutants, and SDSL EPR spectroscopy, we show the octomeric assembly of HbpS in solution, and demonstrate that iron-mediated stress induces conformational changes in HbpS subunits within the octamer. We further demonstrate that the catalase-peroxidase CpeB protects HbpS from hydrogen peroxide (H(2)O(2))-mediated oxidative attack in vivo. Moreover, chromosomal inactivation of cpeB results in an enhanced sensitivity of the mutant to redox-cycling compounds.
  
  
  METHODS
  SDSL EPR has been used in this work for the first time to monitor redox-mediated conformational changes in a redox-sensing protein in solution. This work substantially explains redox-dependent dynamics in HbpS at the atomic level, and presents novel molecular mechanisms supporting downregulation of a signaling cascade.
  
  
  CONCLUSIONS
  Iron-mediated stress induces movements of subunits within the HbpS octomeric assembly. We suggest a motion of the C-terminal α-helix toward the preceding helical segment. These events upregulate the activity of the HbpS-SenS-SenR system, in which HbpS acts as an accessory element. The mycelia-associated CpeB, under the control of HbpS-SenS-SenR, protects the extracellular HbpS from oxidation in vivo. Thus, de novo synthesized HbpS proteins downregulate the HbpS-SenS-SenR signaling cascade.
authors.affiliation: Department of Physics, University of Osnabrueck, Osnabrueck, Germany.
authors.forename: Johann P
authors.initials: JP
authors.lastname: Klare
authors.forename: Darío
authors.initials: D
authors.lastname: Ortiz de Orué Lucana
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002374:Catalase / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D015398:Signal Transduction; D013113:Spin Labels; D013302:Streptomyces / Q000201:enzymology
chemical_list: D002352:Carrier Proteins; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D013113:Spin Labels; D010544:Peroxidases; D002374:Catalase