Catalysis and oxygen binding of Ec DOS: a haem-based oxygen-sensor enzyme from Escherichia coli

Abstract

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.

Identification and Functional and Spectral Characterization of a Globin-coupled Histidine Kinase from Anaeromyxobacter sp. Fw109-5

“…The dissociation rate constant of CO for AfGcHK was significantly lower than those (0.019 -0.067 s Ϫ1 ) of the other oxygen sensor enzymes and SWMb (45). The equilibrium dissociation constant (0.081 M) of CO, calculated from the association and dissociation rate constants of CO for AfGcHK, was similar to those (0.037-0.20 M for: YddV, HemAT-Bs, BpeGReg, and SWMb) of the globin proteins (28,31,33,45) but substantially lower than those of heme-bound PAS proteins (3.1-9.0 M for EcDOS and BjFixL) (42,43,46).…”

“…Stability (42,43,46). AfGcHK may need to detect the presence of trace amounts of oxygen in the bacterial anaerobic environment to switch catalysis, whereas PAS oxygen sensor enzymes are required to detect lowering of the oxygen concentration under normoxia conditions (260 M or saturated O 2 concentration, 1.3 mM) in an aerobic bacterial environment.…”

“…S3) (28, 40 -42). The second-order rate constant for O 2 association with full-length AfGcHK was composed of a two phases with rate constants of 1.3 and 0.15 M Ϫ1 s Ϫ1 at a ϳ1:1 ratio ( (42,43,46) and lower than those (7-32…”

Phosphodiesterase DosP increases persistence by reducing cAMP which reduces the signal indole

“…The heme binding domain of DosP is 60% homologous to the PAS oxygen sensing domain of FixL, an oxygen responsive biological sensor in rhizobia (Delgado‐Nixon et al, ). The catalytic activity of DosP is therefore oxygen dependent (Kobayashi et al, ) and is enhanced 17 fold when saturated with O 2 (Tuckerman et al, ). DosP is also activated during entry into stationary phase and is positively regulated by RpoS, the stationary phase sigma factor (Sommerfeldt et al, ).…”

The Heme-Based Oxygen-Sensor Phosphodiesterase Ec DOS (DosP): Structure-Function Relationships

“…Pulse radiolysis is a powerful tool for investigating electron transfer within proteins, often allowing an electron to be introduced rapidly and selectively into one redox center of an enzyme [ 65 ]. Binding of the O 2 ligand following reduction of the heme iron complex in dimeric Ec DOS-PAS-A-heme Fe(III) was examined using pulse radiolysis [ 66 ]. The kinetics of O 2 ligation to the heme Fe(II) complex in the dimer was a two-phase process reflecting the stepwise reduction of two heme complexes upon application of a high-dose pulse.…”