NO synthase (NOS) is a family of enzymes that catalyzes the NADPH-dependent formation of NO and citrulline from L-arginine and molecular oxygen. The reaction involves an initial hydroxylation of L-arginine to form the isolable intermediate NG-hydroxy-L-arginine (NOHArg). The subsequent incorporation of a second atom of oxygen during the metabolism of NOHArg is required to yield the final products NO and citrulline. NOS contains heme iron, FAD, FMN, and tetrahydrobiopterin prosthetic groups. To examine the interaction of substrates with the heme prosthetic group, substrate perturbation difference spectrophotometry was employed. By analogy with substrate binding interactions with cytochromes P450, NOS exhibits "type I" substrate perturbation difference spectra with the substrates L-arginine and NOHArg and the inhibitor NG-methyl-L-arginine (NMA). These spectral perturbations are characterized by the appearance in the difference spectrum of a peak at approximately 380 nm, a trough with an absorbance minimum at approximately 420 nm, and an isosbestic point at approximately 405 nm. The spectral binding constants, Ks, for L-arginine and NMA were determined to be approximately 2.5 microM. These values are in agreement with the reported kinetic constants for these compounds. The "apparent Ks" values for NOHArg were 0.4 microM (2.0 microM NOS) and 0.8 microM (3.5 microM NOS), respectively. Furthermore, NOS exhibits "type II" difference spectra upon titration with imidazole, characterized by the appearance of a peak at approximately 430 nm and a trough at approximately 395 nm, with a spectral binding constant of approximately 160 microM.