Nitric oxide synthase catalyzes the pyridine nucleotide-dependentoxidation of
L-arginine tonitric oxide and
L-citrulline. It is a specializedcytochrome P
450 monooxygenase that is sensitive toinhibitionby imidazole. Steady-state kinetic studies on recombinant humaninducible nitric oxide synthase (rH-iNOS) demonstrate that imidazole and 1-phenylimidazole are competitiveand reversible inhibitors versus
L-arginine. Structure-activity relationship and pHdependence studies on the inhibition suggest that theneutral form of imidazole may be the preferred species and that theonly modifications allowed withoutthe 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 differencespectra exhibiting
Kd values of 63± 2 and 28 ± 3
M, respectively. These values were in goodagreement with the steady-state
Ki of95± 10 and 38 ± 4
M, respectively, and confirms the site ofbinding is at the sixth axial ligand of theheme. Imidazole (2.2 mM) also perturbed the
Kd of
L-arginine from 3.03 ± 0.45to 209 ± 10
M. Theobserved increase in the
Kd for
L-arginine is consistent with imidazole being a competitiveinhibitor versus
L-arginine. The IC
50 values of imidazoleand 1-phenylimidazole were lower in the absence ofexogenousBH
4, and both inhibitors also competitively inhibited theBH
4-dependent activation of the enzyme.Thesedata taken together suggest that the
L-arginine, dioxygen,and the BH
4 binding sites are in closeproximityin rH-iNOS. Furthermore, these studies demonstrate the usefulnessof imidazole compounds as activesite probes for recombinant human iNOS.