Heme ligand binding properties and intradimer interactions in the full-length sensor protein dos from Escherichia coli and its isolated heme domain.
Dos from Escherichia coli is a bacterial gas sensor protein comprising a heme-containing gas sensor domain and a phosphodiesterase catalytic domain. Using a combination of static light scattering and gel filtration experiments, we established that, as are many other sensor proteins, the full-length protein is dimeric. The full-length dimer (association constant <10 nm) is more stable than the dimeric heme domain (association constant approximately 1 mum), and the dimer interface presumably includes both sensor and catalytic domains. Ultrafast spectroscopic studies showed little influence of the catalytic domain on kinetic processes in the direct vicinity of the heme. By contrast, the properties of ligand (CO and O(2)) binding to the heme in the sensor domain, occurring on a microsecond to second time scale, were found to be influenced by (i) the presence of the catalytic domain, (ii) the dimerization state, and in dimers, (iii) the ligation state of the other subunit. These results imply allosteric interactions within dimers. Steady-state titrations demonstrated marked cooperativity in oxygen binding to both the full-length protein and the isolated heme domain, a feature not reported to date for any dimeric sensor protein. Analysis of a variety of time-resolved experiments showed that Met-95 plays a major role in the intradimer interactions. The intrinsic binding and dissociation rates of Met-95 to the heme were modulated approximately 10-fold by intradimer and sensor-catalytic domain interactions. Dimerization effects were also observed for cyanide binding to the ferric heme domains, suggesting a similar role for Met-95 in ferric proteins.
INSERM U779, Universités Paris VI et XI, 94276 Le Kremlin-Bicêtre, France.
Christophe
C
Lechauve
Laboratory of Optics and Biosciences, CNRS, Ecole Polytechnique, 91128 Palaiseau, France; INSERM U696, 91228 Palaiseau, France.
Latifa
L
Bouzhir-Sima
Laboratory of Optics and Biosciences, CNRS, Ecole Polytechnique, 91128 Palaiseau, France; INSERM U696, 91228 Palaiseau, France.
Taku
T
Yamashita
INSERM U779, Universités Paris VI et XI, 94276 Le Kremlin-Bicêtre, France.
Michael C
MC
Marden
Laboratory of Optics and Biosciences, CNRS, Ecole Polytechnique, 91128 Palaiseau, France; INSERM U696, 91228 Palaiseau, France. Electronic address: marten.vos@polytechnique.edu.
Marten H
MH
Vos
Laboratory of Optics and Biosciences, CNRS, Ecole Polytechnique, 91128 Palaiseau, France; INSERM U696, 91228 Palaiseau, France.
Ursula
U
Liebl
INSERM U779, Universités Paris VI et XI, 94276 Le Kremlin-Bicêtre, France. Electronic address: Laurent.Kiger@inserm.fr.
Laurent
L
Kiger
D002248:Carbon Monoxide / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D055503:Protein Multimerization / Q000502:physiology*; D020836:Protein Structure, Quaternary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology
D029968:Escherichia coli Proteins; D008024:Ligands; D006418:Heme; D002248:Carbon Monoxide; D010727:Phosphoric Diester Hydrolases; C464427:dosP protein, E coli; D010100:Oxygen