title: Characterization of a direct oxygen sensor heme protein from Escherichia coli. Effects of the heme redox states and mutations at the heme-binding site on catalysis and structure.
abstract: A protein containing a heme-binding PAS (PAS is from the protein names in which imperfect repeat sequences were first recognized: PER, ARNT, and SIM) domain from Escherichia coli has been implied a direct oxygen sensor (Ec DOS) enzyme. In the present study, we isolated cDNA for the Ec DOS full-length protein, expressed it in E. coli, and examined its structure-function relationships for the first time. Ec DOS was found to be tetrameric and was obtained as a 6-coordinate low spin ferric heme complex. Its alpha-helix content was calculated as 53% by CD spectroscopy. The redox potential of the heme was found to be +67 mV versus SHE. Mutation of His-77 of the isolated PAS domain abolished heme binding, whereas mutation of His-83 did not, suggesting that His-77 is one of the heme axial ligands. Ferrous, but not ferric, Ec DOS had phosphodiesterase (PDE) activity of nearly 0.15 min(-1) with cAMP, which was optimal at pH 8.5 in the presence of Mg(2+) and was strongly inhibited by CO, NO, and etazolate, a selective cAMP PDE inhibitor. Absorption spectral changes indicated tight CO and NO bindings to the ferrous heme. Therefore, the present study unequivocally indicates for the first time that Ec DOS exhibits PDE activity with cAMP and that this is regulated by the heme redox state.
authors.affiliation: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan.
authors.forename: Yukie
authors.initials: Y
authors.lastname: Sasakura
authors.forename: Satoshi
authors.initials: S
authors.lastname: Hirata
authors.forename: Shunpei
authors.initials: S
authors.lastname: Sugiyama
authors.forename: Shingo
authors.initials: S
authors.lastname: Suzuki
authors.forename: Sue
authors.initials: S
authors.lastname: Taguchi
authors.forename: Miki
authors.initials: M
authors.lastname: Watanabe
authors.forename: Toshitaka
authors.initials: T
authors.lastname: Matsui
authors.forename: Ikuko
authors.initials: I
authors.lastname: Sagami
authors.forename: Toru
authors.initials: T
authors.lastname: Shimizu
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D015374:Biosensing Techniques; D002384:Catalysis; D002942:Circular Dichroism; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000032:analysis*; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D006420:Hemeproteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D010100:Oxygen