| Abstract
| - The cytokine-inducible NO synthase (iNOS) is a flavin-containinghemeprotein that mustdimerize to generate NO. Trypsin cleaves the dimeric enzyme intoan oxygenase domain fragment thatremains dimeric, contains heme and H4biopterin, and bindsl-arginine and a reductase domain fragmentthat is monomeric, binds NADPH, FAD, FMN, and catalyzes the reductionof cytochrome c [Ghosh, D.K. & Stuehr, D. J. (1995) Biochemistry34,801−807]. The current study investigates the isolatedoxygenaseand reductase domains of iNOS to understand how they form and stabilizethe active dimeric enzyme.The dimeric oxygenase domain dissociated into folded,heme-containing monomers when incubated with2−5 M urea, whereas the reductase domain unfolded under theseconditions and lost its ability to catalyzeNADPH-dependent cytochrome c reduction. Spectralanalysis of the dissociation reaction showed that itcaused structural changes within the oxygenase domain and exposed thedistal side of the heme to solvent,enabling it to bind dithiothreitol as a sixth ligand. Importantly,the oxygenase domain monomers couldreassociate into a dimeric form even in the absence of the reductasedomain. The reaction requiredl-arginine and H4biopterin and completely reversed thestructural changes in heme pocket and proteinstructure that occurred upon dissociating the original dimer.Together, this confirms that the oxygenasedomain contains all of the determinants needed for subunit dimerizationand indicates that the dimericstructure greatly affects the heme and protein environment in theoxygenase domain.
|
