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.

Critical roles of Leu99 and Leu115 at the heme distal side in auto-oxidation and the redox potential of a heme-regulated phosphodiesterase from Escherichia coli.

The heme-regulated phosphodiesterase from Escherichia coli (Ec DOS), which is a heme redox-dependent enzyme, is active with a ferrous heme but inactive with a ferric heme. Global structural changes including axial ligand switching and a change in the rigidity of the FG loop accompanying the heme redox change may be related to the dependence of Ec DOS activity on the redox state. Axial ligands such as CO, NO, and O2 act as inhibitors of Ec DOS because they interact with the ferrous heme complex. The X-ray crystal structure of the isolated heme-bound domain (Ec DosH) shows that Leu99, Phe113 and Leu115 indirectly and directly form a hydrophobic triad on the heme plane and that they should be located at or near the ligand access channel of the heme iron. We generated L99T, L99F, L115T, and L115F mutants of Ec DosH and examined their physicochemical characteristics, including auto-oxidation rates, O2 and CO binding kinetics, and redox potentials. The Fe(III) complex of the L115F mutant was unstable and had a Soret absorption spectrum located 5 nm lower than those of the wild-type and other mutants. Auto-oxidation rates of the mutants (0.049-0.33 min(-1)) were much higher than that of the wild-type (0.0063 min(-1)). Furthermore, the redox potentials of the former three mutants (23.1-34.6 mV versus SHE) were also significantly lower than that of the wild-type (63.9 mV versus SHE). Interaction between O2 and the L99F mutant was different from that in the wild-type, whereas CO binding rates of the mutants were similar to those of the wild-type. Thus, it appears that Leu99 and Leu115 are critical for determining the characteristics of heme iron. Finally, we discuss the roles of these amino-acid residues in the heme electronic states.

DOS(Ec), a heme-regulated phosphodiesterase, plays an important role in the regulation of the cyclic AMP level in Escherichia coli.

Heme-regulated phosphodiesterase from Escherichia coli (DOS(Ec)) catalyzes the hydrolysis of cyclic AMP (cAMP) in vitro and is regulated by the redox state of the bound heme. Changes in the redox state result in alterations in the three-dimensional structure of the enzyme, which is then transmitted to the functional domain to switch catalysis on or off. Because DOS(Ec) was originally cloned from E. coli genomic DNA, it has not been known whether it is actually expressed in wild-type E. coli. In addition, the turnover number of DOS(Ec) using cAMP as a substrate is only 0.15 min(-1), which is relatively low for a physiologically relevant enzyme. In the present study, we demonstrated for the first time that the DOS(Ec) gene and protein are expressed in wild-type E. coli, especially under aerobic conditions. We also developed a DOS(Ec) gene knockout strain (Deltados). Interestingly, the knockout of dos caused excess accumulation of intracellular cAMP (26-fold higher than in the wild-type strain) under aerobic conditions, whereas accumulation of cAMP was not observed under anaerobic conditions. We also found differences in cell morphology and growth rate between the mutant cells and the wild-type strain. The changes in the knockout strain were partially complemented by introducing an expression plasmid for dos. Thus, the present study revealed that expression of DOS(Ec) is regulated according to environmental O2 availability at the transcriptional level and that the concentration of cAMP in cells is regulated by DOS(Ec) expression.