Macrophage nitric oxide synthase subunits. Purification, characterization, and role of prosthetic groups and substrate in regulating their association into a dimeric enzyme

J Biol Chem. 1993 Oct 5;268(28):21120-9.

Abstract

The cytokine-induced nitric oxide synthase (NOS) of macrophages is a homodimeric enzyme that contains iron protoporphorin IX (heme), FAD, FMN, tetrahydrobiopterin, and calmodulin. To investigate how the enzyme's quaternary structure relates to its catalytic activity and binding of prosthetic groups, dimeric NOS and its subunits were purified separately and their composition and catalytic properties compared. In contrast to dimeric NOS, purified subunits did not synthesize NO or contain bound heme or tetrahydrobiopterin. However, the subunits did contain FAD, FMN, and calmodulin in amounts comparable with dimeric NOS, displayed the light absorbance spectrum of an FAD- and FMN-containing flavoprotein, and generated an air-stable flavin semiquinone radical upon reduction of their ferricyanide-oxidized form. Dimeric NOS and NOS subunits were equivalent in catalyzing electron transfer from NADPH to cytochrome c, dichlorophenolindophenol, or ferricyanide at rates that were 8-30-fold faster than the maximal rate of NO synthesis by dimeric NOS. Reconstitution of subunit NO synthesis required their incubation with L-arginine, tetrahydrobiopterin, and stoichiometric amounts of heme and correlated with formation of a proportional amount of dimeric NOS in all cases. The dimeric NOS reconstituted from its subunits contained 0.9 heme and 0.44 tetrahydrobiopterin bound per subunit and had the spectral and catalytic properties of native dimeric NOS. Thus, NOS subunits are NADPH-dependent reductases that acquire the capacity to synthesize NO only through their dimerization and binding of heme and tetrahydrobiopterin. The ability of heme, tetrahydrobiopterin, and L-arginine to promote subunit dimerization is unprecedented and suggests novel roles for these molecules in forming and stabilizing the active dimeric NOS.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Oxidoreductases / chemistry
  • Amino Acid Oxidoreductases / isolation & purification
  • Amino Acid Oxidoreductases / metabolism*
  • Animals
  • Biopterin / analogs & derivatives
  • Biopterin / metabolism
  • Catalysis
  • Chromatography, Gel
  • Electrophoresis, Polyacrylamide Gel
  • Flavin Mononucleotide / metabolism
  • Flavin-Adenine Dinucleotide / metabolism
  • Heme / metabolism
  • Macrophages / enzymology*
  • Mice
  • NADP / metabolism
  • Nitric Oxide / biosynthesis
  • Nitric Oxide Synthase
  • Oxidation-Reduction
  • Oxidoreductases / metabolism
  • Substrate Specificity

Substances

  • Flavin-Adenine Dinucleotide
  • Biopterin
  • Nitric Oxide
  • Heme
  • NADP
  • Flavin Mononucleotide
  • Oxidoreductases
  • Nitric Oxide Synthase
  • Amino Acid Oxidoreductases
  • sapropterin