Understanding the structural aspects of neuronal nitric oxide synthase (NOS) using microdissection by molecular cloning techniques: molecular dissection of neuronal NOS.

Abstract

The neuronal isoform (Type I) of nitric oxide synthase (NOS) requires Ca+2/calmodulin to catalyze the formation of NO● and citrulline from L-arginine (1) and molecular oxygen (2) with reducing equivalents from NADPH. The overall reaction is a five-electron process involving two successive monooxygenation steps with the obligatory formation of N-hydroxy-L-arginine as the oxygenated intermediate. The neuronal NOS shares with the other two known isoforms the unusual property, for a mammalian enzyme, of containing iron protoporphyrin IX (3–6), FAD and FMN (3, 7–9), and tetrahydrobiopterin (9). Bredt, et al. (10) had previously demonstrated the remarkable (58%) sequence similarity of rat brain NOS to rat liver NADPH-cytochrome P450 reductase and determined that the C-terminal 641 amino acids of NOS display consensus regions for both flavin and nucleotide binding. It was reported that carbon monoxide (CO) inhibited both macrophage and neuronal NOS activity (3–6) and these various preparations exhibited reduced-CO difference spectra with absorbance maxima in the region of 445 nm, a property shared with the members of the cytochrome P450 family. However, the low degree of sequence similarity to any of the more than 200 members of this family and other CO-binding, oxygenating heme proteins, such as chloroperoxidase or Bacillus megaterium P450 (BM-3), suggests that the homology may end with the sharing of a cysteine thiolate ligand to the heme iron at the fifth axial position. Recent studies have established that this involves cysteine415 of the neuronal NOS (11, 12), as suggested originally by McMillan, et al. (3), and cysteine184 of the endothelial NOS (13) as determined by site-directed mutagenesis of the holoenzyme (11,13) and the heme domain (12), respectively.