DOMAINS OF MACROPHAGE NO SYNTHASE HAVE DIVERGENT ROLES IN FORMING ANDSTABILIZING THE ACTIVE DIMERIC ENZYME

The cytokine-inducible NO synthase (iNOS) is a flavin-containing hemep rotein that must dimerize to generate NO. Trypsin cleaves the dimeric enzyme into an oxygenase domain fragment that remains dimeric, contain s heme and H4biopterin, and binds L-arginine and a reductase domain fr agment that is monomeric, binds NADPH, FAD, FMN, and catalyzes the red uction of cytochrome c [Ghosh, D. K. & Stuehr, D. J. (1995) Biochemist ry 34, 801-807]. The current study investigates the isolated oxygenase and reductase domains of iNOS to understand how they form and stabili ze the active dimeric enzyme. The dimeric oxygenase domain dissociated into folded, heme-containing monomers when incubated with 2-5 M urea, whereas the reductase domain unfolded under these conditions and lost its ability to catalyze NADPH-dependent cytochrome c reduction. Spect ral analysis of the dissociation reaction showed that it caused struct ural changes within the oxygenase domain and exposed the distal side o f the heme to solvent, enabling it to bind dithiothreitol as a sixth l igand. importantly, the oxygenase domain monomers could reassociate in to a dimeric form even in the absence of the reductase domain. The rea ction required L-arginine and H4biopterin and completely reversed the structural changes in heme pocket and protein structure that occurred upon dissociating the original dimer. Together, this confirms that the oxygenase domain contains all of the determinants needed for subunit dimerization and indicates that the dimeric structure greatly affects the heme and protein environment in the oxygenase domain.