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.

Arg97 at the heme-distal side of the isolated heme-bound PAS domain of a heme-based oxygen sensor from Escherichia coli (Ec DOS) plays critical roles in autoxidation and binding to gases, particularly O2.

The catalytic activity of heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) on cyclic di-GMP is markedly enhanced upon binding of gas molecules, such as O2 and CO, to the heme iron complex in the sensor domain. Arg97 interacts directly with O2 bound to Fe(II) heme in the crystal structure of the isolated heme-bound sensor domain with the PAS structure (Ec DOS-PAS) and may thus be critical in ligand recognition. To establish the specific role of Arg97, we generated Arg97Ala, Arg97Glu, and Arg97Ile mutant Ec DOS-PAS proteins and examined binding to O2, CO, and cyanide, as well as redox potentials. The autoxidation rates of the Arg97Ala and Arg97Glu mutant proteins were up to 2000-fold higher, while the O2 dissociation rate constant for dissociation from the Fe(II)-O2 heme complex of the Arg97Ile mutant was 100-fold higher than that of the wild-type protein. In contrast, the redox potential values of the mutant proteins were only slightly different from that of the wild type (within 10 mV). Accordingly, we propose that Arg97 plays critical roles in recognition of the O2 molecule and redox switching by stabilizing the Fe(II)-O2 complex, thereby anchoring O2 to the heme iron and lowering the autoxidation rate to prevent formation of Fe(III) hemin species not regulated by gas molecules. Arg97 mutations significantly influenced interactions with the internal ligand Met95, during CO binding to the Fe(II) complex. Moreover, the binding behavior of cyanide to the Fe(III) complexes of the Arg mutant proteins was similar to that of O2, which is evident from the Kd values, suggestive of electrostatic interactions between cyanide and Arg97.