Roles of Arg-97 and Phe-113 in Regulation of Distal Ligand Binding to Heme in the Sensor Domain of Ec DOS Protein

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 3 Ile, Met-95 3 Ala, Arg-97 3 Ile, Arg-97 3 Ala, Arg-97 3 Glu, Phe-113 3 Leu, and Phe-113 3 Thr) of the heme-containing PAS domain of Ec DOS. For the CO-and NO-bound forms, both the hydrogen-bonded and nonhydrogen-bonded conformations were found, and in the former Heme-based sensors are a class of enzymes that regulates the enzymatic activities and DNA binding in response to the presence of diatomic gas molecules CO, NO, or O 2 (1-6). The direct oxygen sensor from Escherichia coli (Ec DOS) 4 is a hemebased signal transducer protein responsible for phosphodiesterase (PDE) activity (7,8). The Ec DOS is composed of a C-terminal PDE catalytic domain and the N-terminal hemecontaining domain. The latter is a prototypical PAS domain, which is a ubiquitous protein sensory domain found in all kingdoms (9), and has a conserved ␣/␤ folds consisting of ϳ147 residues (10, 11). The Ec DOS protein has a PDE activity specific to 3Ј,5Ј-cyclic diguanylic acid (12, 13), and the binding of O 2 , CO, or NO to the reduced heme significantly enhances the PDE activity (14, 15). Thus, Ec DOS is a novel gas-sensor enzyme that has unprecedented ability to be activated by different gas molecules. Because the CO and NO concentrations in the cells are very low (nanomolar), it is likely that Ec DOS is predominantly an oxygen sensor enzyme whereby catalysis is regulated in response to the micromolar O 2 level (16). It is believed that the structural changes in the heme vicinity caused by ligand binding to the heme provide the initial event in the gas sensing, followed by intramolecular signal transduction from the heme to the functional domain, and thus regulating the PDE activity. Fig. 1 illustrates the crystal structure of the truncated heme sensor domain (Ec DOSH) of Ec DOS protein (10). In the reduced form (Fig. 1A), Met-95 and His-77 are the heme axial ligands in the distal and proximal sides, respectively, and Met-95 forms a hydrogen bond with heme 7-propionate. Upon O 2 binding to the reduced heme, Met-95 is replaced by O 2 and heme 7-propionate forms a hydrogen bond with Arg-97 instead of Met-95. This stabilizes the heme-coordinated O 2 (Fig. 1B). Thus, the replacement of a distal axial ligand from Met-95 to O 2 perturbs the heme 7-propionate hydrogen bonding network, resulting in large conformational changes in the FG loop (10). On the other hand, the role of other distal residues (Fig. 1) and the heme 7-propionate hydrogen bonding network are not clear in the CO-or NO-sensing mechanism.To understand the gas-sensing mechanism of Ec DOS, it is essential to determine the changes in the heme and surrounding structures caused by distal ligand binding/di...

Dynamics of Methionine Ligand Rebinding in Cytochrome c

“…Although heme−ligand dissociation in heme proteins usually does not occur under normal conditions, heme−ligand bond breaking and rebinding take place during the process of internal and external ligand switching or competition in neuroglobin 30,31 as well as in a specific group of heme-based sensor proteins such as Ec DOS, DOSH, and CooA. 7,32,33 Understanding the kinetics and thermodynamics of internal ligand binding is essential to our knowledge of the composition and function of these heme proteins.Cytochrome c (cyt c) (Figure 1) is an essential component of the electron transport chain in mitochondria. 34 The 6-coordinated iron (Fe) in the heme plays an essential role in the process of electron transport.…”

Role of Phe113 at the distal side of the heme domain of an oxygen-sensor (Ec DOS) in the characterization of the heme environment

“…Thus, we hypothesized that the hydrophobic amino acid residue at position 113 might contribute to recognition of the O 2 molecule. A resonance Raman spectral study supported this idea, in that significant interactions of F113 with CO bound to the Fe(II) complex occurred upon photo-dissociation of CO [8].…”

“…The M95 ligation to the Fe(II) complex seen in difference spectra in the 100-ls time domain was also suggested by transient resonance Raman spectra [8]. Mutations at residue 113 did not significantly change the rate of M95 ligation (the first phase, Table S2 of Supplementary material).…”

“…Thus, it is suggested that the CO access channel to the Fe(II) complex of the F113 mutants is changed in a manner facilitating CO binding. The stretching mode frequency (496 cm À1 ) corresponding to the Fe-CO bond of the F113L mutant in resonance Raman spectra differed from the frequencies (486-488 cm À1 ) of the wild-type, and other mutant proteins [8].…”

“…Cloning of Ec DOS-PAS and construction of the expression plasmid, pET28a(+), containing cDNA encoding Ec DOS-PAS, were performed as described previously [3,5,[7][8][9][10][11].…”