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.
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