Catalysis and oxygen binding of Ec DOS: a haem-based oxygen-sensor enzyme from Escherichia coli.
A phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is a novel haem-based oxygen sensor enzyme. Binding of O(2) to the reduced haem in the sensor domain enhances PDE activity exerted by the catalytic domain. Kinetic analysis of oxygen-dependent catalytic enhancement showed a sigmoidal curve with a Hill coefficient value of 2.8. To establish the molecular mechanism underlying allosteric regulation, we analysed binding of the O(2) ligand following reduction of haem in the isolated dimeric sensor domain using pulse radiolysis. Spectral changes accompanying O(2) binding were composed of two phases as a result of reduction of two haem complexes when high-dose electron beams were applied. In contrast, upon reduction of the dimer with a low-dose beam, the kinetics of O(2) ligation displayed single-phase behaviour as a result of the reduction of one haem complex within dimer. Based on these results, we propose that the faster phase corresponds to binding of the first O(2) molecule to one subunit of the dimer, followed by binding of the second O(2) molecule to the other subunit. Notably, for the haem axial ligand mutant proteins, M95A and M95L, O(2) binding displayed single-phase kinetics and was independent of electron beam dose.
Kobayashi
Kazuo
K
The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka, Japan. kobayasi@sanken.osaka-u.ac.jp
Tanaka
Atsunari
A
Takahashi
Hiroto
H
Igarashi
Jotaro
J
Ishitsuka
Yukako
Y
Yokota
Nao
N
Shimizu
Toru
T
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization / Q000502:physiology; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011675:Pulse Radiolysis
D001426:Bacterial Proteins; D008024:Ligands; D021122:Protein Subunits; D006418:Heme; D010727:Phosphoric Diester Hydrolases; D010100:Oxygen