title: Unusual heme binding properties of the dissimilative nitrate respiration regulator, a bacterial nitric oxide sensor.
abstract: OBJECTIVE
  In the opportunistic pathogen Pseudomonas aeruginosa, nitric oxide (NO) triggers the respiration of nitrate (denitrification), thus allowing survival in chronic infection sites as a microaerobic-anaerobic biofilm. The NO-dependent induction of denitrification is mediated by the dissimilative nitrate respiration regulator (DNR), a transcription factor forming a stable complex with heme, which is required to sense the physiological messenger (i.e., NO). The molecular details of NO sensing in DNR and, more in general, in this class of sensors are largely unknown, and a study aimed at integrating microbiology and biochemistry is needed.
  
  
  RESULTS
  Here we present a comprehensive study, including in vivo results and spectroscopy, kinetics, and protein engineering, that demonstrates the direct involvement of a histidine residue in heme iron coordination. Moreover, a peculiar phenomenon of ligand switching around heme iron, which hampers the identification of the second heme axial ligand, is also suggested. These results indicate that DNR is characterized by a remarkable flexibility in solution, as observed for other cAMP receptor protein/fumarate and nitrate reductase regulators (CRP-FNR) to which DNR belongs.
  
  
  METHODS
  The present work represents one of the few studies focused on the biochemistry of NO sensing by bacterial transcriptional regulators. The data presented demonstrate that structural plasticity of DNR is crucial for the sensing activity and confers to the protein unusual heme binding properties.
  
  
  CONCLUSIONS
  Protein flexibility and dynamics is a key structural feature essential to explain the evolutionary success and adaptability of CRP-FNR, and may represent a common strategy employed by heme-based redox sensors, which presents features deeply different from those of canonical hemeproteins.
authors.affiliation: Istituto Pasteur Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Rinaldo
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Castiglione
authors.forename: Giorgio
authors.initials: G
authors.lastname: Giardina
authors.forename: Manuela
authors.initials: M
authors.lastname: Caruso
authors.forename: Alessandro
authors.initials: A
authors.lastname: Arcovito
authors.forename: Stefano Della
authors.initials: SD
authors.lastname: Longa
authors.forename: Paola
authors.initials: P
authors.lastname: D'Angelo
authors.forename: Francesca
authors.initials: F
authors.lastname: Cutruzzolà
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000235:genetics / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D009566:Nitrates; D009569:Nitric Oxide