title: Roles of Arg-97 and Phe-113 in regulation of distal ligand binding to heme in the sensor domain of Ec DOS protein. Resonance Raman and mutation study.
abstract: The direct oxygen sensor protein isolated from Escherichia coli (Ec DOS) is a heme-based signal transducer protein responsible for phosphodiesterase (PDE) activity. Binding of O(2), CO, or NO to a reduced heme significantly enhances the PDE activity toward 3',5'-cyclic diguanylic acid. We report stationary and time-resolved resonance Raman spectra of the wild-type and several mutants (Glu-93 --> Ile, Met-95 --> Ala, Arg-97 --> Ile, Arg-97 --> Ala, Arg-97 --> Glu, Phe-113 --> Leu, and Phe-113 --> Thr) of the heme-containing PAS domain of Ec DOS. For the CO- and NO-bound forms, both the hydrogen-bonded and non-hydrogen-bonded conformations were found, and in the former Arg-97 forms a hydrogen bond with the heme-bound external ligand. The resonance Raman results revealed significant interactions of Arg-97 and Phe-113 with a ligand bound to the sixth coordination site of the heme and profound structural changes in the heme propionates upon dissociation of CO. Mutation of Phe-113 perturbed the PDE activities, and the mutation of Arg-97 and Phe-113 significantly influenced the transient binding of Met-95 to the heme upon photodissociation of CO. This suggests that the electrostatic interaction of Arg-97 and steric interaction of Phe-113 are crucial for regulating the competitive recombination of Met-95 and CO to the heme. On the basis of these results, we propose a model for the role of the heme propionates in communicating the heme structural changes to the protein moiety.
authors.affiliation: Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan.
authors.forename: Samir F
authors.initials: SF
authors.lastname: El-Mashtoly
authors.forename: Satoru
authors.initials: S
authors.lastname: Nakashima
authors.forename: Atsunari
authors.initials: A
authors.lastname: Tanaka
authors.forename: Toru
authors.initials: T
authors.lastname: Shimizu
authors.forename: Teizo
authors.initials: T
authors.lastname: Kitagawa
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases; D017434:Protein Structure, Tertiary / Q000235:genetics; D013059:Spectrum Analysis, Raman
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D009569:Nitric Oxide; D006418:Heme; D010649:Phenylalanine; D002248:Carbon Monoxide; D001120:Arginine; D010727:Phosphoric Diester Hydrolases; C464427:dosP protein, E coli