Optical Difference Spectrophotometry as a Probe of Rat Brain Nitric Oxide Synthase Heme-Substrate Interaction

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 N^G-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 N^G-methyl-l-arginine (NMA). These spectral perturbations are characterized by the appearance in the difference spectrum of a peak at ∼380 nm, a trough with an absorbance minimum at ∼420 nm, and an isosbestic point at ∼405 nm. The spectral binding constants, K_s, for l-arginine and NMA were determined to be ∼2.5 µM. These values are in agreement with the reported kinetic constants for these compounds. The “apparent K_s” values for NOHArg were 0.4 µM (2.0 µM NOS) and 0.8 µM (3.5 µM NOS), respectively. Furthermore, NOS exhibits “type II” difference spectra upon titration with imidazole, characterized by the appearance of a peak at ∼430 nm and a trough at ∼395 nm, with a spectral binding constant of ∼160 µM. The conversion of NOS (∼75% total protein at 1 mM imidazole) to a “low-spin” species suggests the displacement of an unknown bound ligand that interacts with the heme prosthetic group. The high-spin-state species is restored upon addition of l-arginine, independent of the presence of imidazole. d-Arginine, which is neither a substrate nor an inhibitor of NOS, does not exhibit a titratable interaction with the heme. Thus, the substrate l-arginine and the reaction intermediate NOHArg exhibit a specific and titratable interaction with the heme prosthetic group, implicating the heme as the reaction center for the initial binding of substrate and the subsequent incorporation of oxygen to form both products, NO and citrulline.