Binding of oxygen and carbon monoxide to a heme-regulated phosphodiesterase from Escherichia coli: Kinetics and infrared spectra of the full-length wild-type enzyme, isolated PAS domain, and MET-95 mutants

The heme-regulated phosphodiesterase, Ec DOS, is a redox sensor that uses the heme in its PAS domain to regulate catalysis. The rate of O₂ association (k_on) with full-length Ec DOS is extremely slow at 0. 0019 μM^-1 s^-1, compared with >9.5 μM ^-1 s^-1 for 6-coordinated globin-type hemoproteins, as determined by the stopped-flow method. This rate is dramatically increased (up to 16-fold) in the isolated heme-bound PAS domain. Dissociation constants (K_d) calculated from the kinetic parameters are 340 and 20 μM for the full-length wild-type enzyme and its isolated PAS domain, respectively. Mutations at Met-95 in the isolated PAS domain, which may be a heme axial ligand in the Fe(II) complex, lead to a further increase in the k_on value by more than 30-fold, and consequently, a decrease in the K_d value to less than 1 Μ. The k_on value for CO binding to the full-length wild-type enzyme is also very low (0.00081 μM^-1 s ^-1). The kinetics of CO binding to the isolated PAS domain and its mutants are similar to those observed for O₂. However, the K _d values for CO are considerably lower than those for O₂.