Heme iron reduction and catalysis by a nitric oxide synthase heterodimer containing one reductase and two oxygenase domains.
Inducible nitric oxide (NO) synthase (iNOS) is comprised of an oxygenase 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 between two oxygenase domains with the reductase domains attached as monomeric extensions. To understand how dimerization activates iNOS, we synthesized an iNOS heterodimer comprised of one full-length subunit and one histidine-tagged subunit that was missing its reductase domain. The heterodimer was purified using nickel-Sepharose and 2',5'-ADP affinity chromatography. The heterodimer catalyzed NADPH-dependent NO synthesis 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 synthesis 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 iNOS monomer preparations could not synthesize NO 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 can reduce only one of two hemes in a dimer, this supports NO synthesis from L-arginine.
Siddhanta
U
U
Department of Immunology, Cleveland Clinic Research Institute, Ohio 44195, USA.
Wu
C
C
Abu-Soud
H M
HM
Zhang
J
J
Ghosh
D K
DK
Stuehr
D J
DJ
D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008264:Macrophages / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D010105:Oxygenases / Q000737:chemistry / Q000378:metabolism*; D013053:Spectrophotometry
D002147:Calmodulin; D046911:Macromolecular Substances; D005182:Flavin-Adenine Dinucleotide; D009569:Nitric Oxide; D006418:Heme; D009249:NADP; D005486:Flavin Mononucleotide; D007501:Iron; D010088:Oxidoreductases; D010105:Oxygenases; D019001:Nitric Oxide Synthase