Escherichia coli phosphodiesterase (Ec DOS1), a gas sensor enzyme, stimulates phosphodiesterase activity toward cyclic-di-GMP when gas molecules such as CO2, O2, and NO gas are bound to it. There is an enhanced catalytic activity towards cyclic diGMP of 6-7- fold upon binding of the gases mentioned prior to the Fe (II)-protoporphyrin IX complex. Fe (III) complex, in this study was shown to display catalytic activity toward cyclic-di-GMP that is analogous to the Fe (II) complex. Instead of gas stimulated catalysis, external ligands cyanide, and imidazole were bound to the Fe (III) complex. This process is important because the external ligands induce a 10- to- 11-fold increase in catalysis, which is more significant than the 6-to-7.2 fold observed for the gas stimulated Fe (II) complex. The full length Ec DOS enzyme is mainly purified as the Fe (III) form in the absence of reductant from the E. coli overexpression system. The wild type and mutant Ec DOS proteins were overexpressed in and E. coli strain and transformed with the pET28a (+)-Ec DOS expression plasmid containing an N-terminal his tag and a thrombin cleave site. The following protein purification procedures were performed on ice or at 4 °C. E. coli cells frozen at -80 °C were suspended in 50 mM potassium phosphate buffer (pH 7.5) containing 20 mM imidazole, 150 mM NaCl, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride (buffer A), and 0.1 mg/ml lysozyme. The purified proteins were then treated with 1mM potassium ferricyanide for complete oxidation. A continuous assay that was analyzed via optical absorption spectroscopy was used to measure the rapid reactions of ligand association to the heme. The spectral changes of the heme to Ec DOS were monitored after the addition of cyanide and imidazole. When they compared the heme free mutant H77A, it had the same effect as the ligand-stimulated enzyme. Therefore the authors believe the binding of the heme iron complex to the Ec DOS sensor domain is not essential...