HEME IRON REDUCTION AND CATALYSIS BY A NITRIC-OXIDE SYNTHASE HETERODIMER CONTAINING ONE REDUCTASE AND 2 OXYGENASE DOMAINS

Inducible nitric oxide (NO) synthase (iNOS) is comprised of an oxygena se domain containing heme, tetrahydrobiopterin, the substrate binding site, and a reductase domain containing FAD, FMN, calmodulin, and the NADPH binding site, Enzyme activity requires a dimeric interaction bet ween two oxygenase domains with the reductase domains attached as mono meric extensions, To understand how dimerization activates iNOS, we sy nthesized an iNOS heterodimer comprised of one full-length subunit and one histidine-tagged subunit that was missing its reductase domain, T he heterodimer was purified using nickel-Sepharose and 2',5'-ADP affin ity chromatography, The heterodimer catalyzed NADPH-dependent NO synth esis from L-arginine at a rate of 52 +/- 6 nmol of NO/min/nmol of heme , which is half the rate of purified iNOS homodimer, Heterodimer NO sy nthesis was associated with reduction of only half of its heme iron by NADPH, in contrast with near complete heme iron reduction in an iNOS homodimer, Full-length MOS monomer preparations could not synthesize N O nor catalyze NADPH-dependent heme iron reduction. Thus, dimerization activates NO synthesis by enabling electrons to transfer between the reductase and oxygenase domains. Although a single reductase domain ca n reduce only one of two hemes in a dimer, this supports NO synthesis from L-arginine.