Active-Site Structure Analysis of Recombinant Human Inducible Nitric Oxide Synthase Using Imidazole

Nitric oxide synthase catalyzes the pyridine nucleotide-dependent oxidation of l-arginine to nitric oxide and l-citrulline. It is a specialized cytochrome P450 monooxygenase that is sensitive to inhibition by imidazole. Steady-state kinetic studies on recombinant human inducible nitric oxide synthase (rH-iNOS) demonstrate that imidazole and 1-phenylimidazole are competitive and reversible inhibitors versus l-arginine. Structure−activity relationship and pH dependence studies on the inhibition suggest that the neutral form of imidazole may be the preferred species and that the only modifications allowed without the loss of inhibition are at the N-1 position of imidazole. Optical spectrophotometric studies of rH-iNOS with imidazole and 1-phenylimidazole yielded type II difference spectra exhibiting Kd values of 63 ± 2 and 28 ± 3 μM, respectively. These values were in good agreement with the steady-state Ki of 95 ± 10 and 38 ± 4 μM, respectively, and confirms the site of binding is at the sixth axial ligand ...