title: Pressure effects reveal that changes in the redox states of the heme iron complexes in the sensor domains of two heme-based oxygen sensor proteins, EcDOS and YddV, have profound effects on their flexibility.
abstract: The catalytic activity of a heme-based oxygen sensor phosphodiesterase from Escherichia coli (EcDOS) towards cyclic diGMP is regulated by the redox state of the heme iron complex in the enzyme's sensing domain and the association of external ligands with the iron center. Specifically, the Fe(II) complex is more active towards cyclic diGMP than the Fe(III) complex, and its activity is further enhanced by O2 or CO binding. In order to determine how the redox state and coordination of the heme iron atom regulate the catalytic activity of EcDOS, we investigated the flexibility of its isolated N-terminal heme-binding domain (EcDOS-heme) by monitoring its spectral properties at various hydrostatic pressures. The most active form of the heme-containing domain, i.e. the Fe(II)-CO complex, was found to be the least flexible. Conversely, the oxidized Fe(III) forms of EcDOS-heme and its mutants had relatively high flexibilities, which appeared to be linked to the low catalytic activity of the corresponding intact enzymes. These findings corroborate the suggestion, made on the basis of crystallographic data, that there is an inverse relationship between the flexibility of the heme-containing domain of EcDOS and its catalytic activity. The Fe(II)-CO form of the heme domain of a second heme-based oxygen sensor, diguanylate cyclase (YddV), was also found to be quite rigid. Interestingly, the incorporation of a water molecule into the heme complex of YddV caused by mutation of the Leu65 residue reduced the flexibility of this heme domain. Conversely, mutation of the Tyr43 residue increased its flexibility.
authors.affiliation: Institute of Pharmacology, Faculty of Medicine, Palacky University, Olomouc, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Anzenbacher
authors.forename: Stéphane
authors.initials: S
authors.lastname: Marchal
authors.forename: Jan
authors.initials: J
authors.lastname: Palacký
authors.forename: Eva
authors.initials: E
authors.lastname: Anzenbacherová
authors.forename: Thomas
authors.initials: T
authors.lastname: Domaschke
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lange
authors.forename: Toru
authors.initials: T
authors.lastname: Shimizu
authors.forename: Kenichi
authors.initials: K
authors.lastname: Kitanishi
authors.forename: Martin
authors.initials: M
authors.lastname: Stranava
authors.forename: Marie
authors.initials: M
authors.lastname: Stiborová
authors.forename: Marketa
authors.initials: M
authors.lastname: Martinkova
mesh_terms: D002384:Catalysis; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006874:Hydrostatic Pressure; D010084:Oxidation-Reduction; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry*; D019761:Phosphorus-Oxygen Lyases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D013056:Spectrophotometry, Ultraviolet
chemical_list: D029968:Escherichia coli Proteins; D010727:Phosphoric Diester Hydrolases; C464427:dosP protein, E coli; D019761:Phosphorus-Oxygen Lyases; C553251:yddV protein, E coli
keywords: heme-based oxygen sensor; intramolecular catalytic regulation; pressure effects; protein compressibility; protein flexibility