title: Dynamics of the heme-binding bacterial gas-sensing dissimilative nitrate respiration regulator (DNR) and activation barriers for ligand binding and escape.
abstract: DNR (dissimilative nitrate respiration regulator) is a heme-binding transcription factor that is involved in the regulation of denitrification in Pseudomonas aeruginosa. In the ferrous deoxy state, the heme is 6-coordinate; external NO and CO can replace an internal ligand. Using fluorescence anisotropy, we show that high-affinity sequence-specific DNA binding occurs only when the heme is nitrosylated, consistent with the proposed function of DNR as NO sensor and transcriptional activator. This role is moreover supported by the NO "trapping" properties revealed by ultrafast spectroscopy that are similar to those of other heme-based NO sensor proteins. Dissociated CO-heme pairs rebind in an essentially barrierless way. This process competes with migration out of the heme pocket. The latter process is thermally activated (Ea ∼ 7 kJ/mol). This result is compared with other heme proteins, including the homologous CO sensor/transcription factor CooA, variants of the 5-coordinate mycobacterial sensor DosT and the electron transfer protein cytochrome c. This comparison indicates that thermal activation of ligand escape from the heme pocket is specific for systems where an external ligand replaces an internal one. The origin of this finding and possible implications are discussed.
authors.affiliation: Laboratory for Optics and Biosciences, CNRS, Ecole Polytechnique, 91128 Palaiseau, France,; INSERM U696, 91128 Palaiseau, France.
authors.forename: Laura
authors.initials: L
authors.lastname: Lobato
authors.affiliation: Laboratory for Optics and Biosciences, CNRS, Ecole Polytechnique, 91128 Palaiseau, France,; INSERM U696, 91128 Palaiseau, France.
authors.forename: Latifa
authors.initials: L
authors.lastname: Bouzhir-Sima
authors.affiliation: Laboratory of Analytical Chemistry, Graduate School of Pharmaceutical Sciences, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan, and.
authors.forename: Taku
authors.initials: T
authors.lastname: Yamashita
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester C04 3SQ, United Kingdom.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Wilson
authors.affiliation: Laboratory for Optics and Biosciences, CNRS, Ecole Polytechnique, 91128 Palaiseau, France,; INSERM U696, 91128 Palaiseau, France,. Electronic address: marten.vos@polytechnique.edu.
authors.forename: Marten H
authors.initials: MH
authors.lastname: Vos
authors.affiliation: Laboratory for Optics and Biosciences, CNRS, Ecole Polytechnique, 91128 Palaiseau, France,; INSERM U696, 91128 Palaiseau, France,. Electronic address: ursula.liebl@polytechnique.edu.
authors.forename: Ursula
authors.initials: U
authors.lastname: Liebl
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology; D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry; D045304:Cytochromes c / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry; D005454:Fluorescence Polarization; D015964:Gene Expression Regulation, Bacterial; D006418:Heme / Q000031:analogs & derivatives / Q000737:chemistry; D006736:Horses; D007700:Kinetics; D008024:Ligands; D009569:Nitric Oxide / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry* / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; C095475:DNR protein, Pseudomonas aeruginosa; D008024:Ligands; D014157:Transcription Factors; D009569:Nitric Oxide; D006418:Heme; C027417:nitrosylprotoheme; D002248:Carbon Monoxide; D045304:Cytochromes c
keywords: Carbon Monoxide; DNA Binding Protein; Energetics; Fluorescence Anisotropy; Heme-based Sensor Protein; Nitric Oxide; Transcription Factor; Ultrafast Spectroscopy