Macrophage NO synthase: characterization of isolated oxygenase and reductase domains reveals a head-to-head subunit interaction.
Macrophage NO synthase (NOS) is a dimeric enzyme comprising two identical 130 kDa subunits and contains iron protoporphyrin IX (heme), tetrahydrobiopterin, FAD, FMN, and calmodulin. We have carried out limited proteolysis to locate the domains involved in prosthetic group binding and subunit interaction. Trypsin cleaved the subunits of dimeric macrophage NOS at a single locus, splitting the enzyme into two fragments whose denatured molecular masses were 56 and 74 kDa. The smaller fragments remained dimeric in their native form (112 kDa), contained heme and tetrahydrobiopterin, and could bind L-arginine, CO, or imidazole. In contrast, the larger fragments were monomeric in their native form, contained FAD, FMN, and CAM, and bound NADPH. Although neither purified fragment alone or in combination catalyzed NO synthesis from L-arginine, the flavin-containing fragment did catalyze cytochrome c reduction at a rate that was equivalent to that of native dimeric NOS. These results indicate that trypsin cuts macrophage NOS into two domains that can exist and function independently of one another. The domain that binds heme, H4biopterin, and substrate is also responsible for maintaining the NOS dimeric structure, while the domain containing FAD, FMN, and CAM is not required for subunit interaction. This suggests a structural model for macrophage NOS in which the subunits align in a head-to-head manner, with the oxygenase domains interacting to form a dimer and the reductase domains existing as independent extensions.
Ghosh
D K
DK
Department of Immunology, Cleveland Clinic, Ohio 44195.
Stuehr
D J
DJ
D000594:Amino Acid Oxidoreductases / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019001:Nitric Oxide Synthase; D010446:Peptide Fragments; D011487:Protein Conformation; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000494:pharmacology
D002147:Calmodulin; D010446:Peptide Fragments; D005182:Flavin-Adenine Dinucleotide; D001708:Biopterins; D006418:Heme; D005486:Flavin Mononucleotide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D014357:Trypsin; C003402:sapropterin