Inhibition of nitric oxide synthase isoforms by tris-malonyl-C(60)-fullerene adducts

Arch Biochem Biophys. 2000 Jun 15;378(2):216-23. doi: 10.1006/abbi.2000.1843.


C(3)-tris-malonyl-C(60)-fullerene and D(3)-tris-malonyl-C(60)-fullerene derivatives inhibit citrulline and NO formation by all three nitric oxide synthase isoforms in a manner fully reversible by dilution. The inhibition of citrulline formation by C(3)-tris-malonyl-C(60)-fullerene occurs with IC(50) values of 24, 17, and 123 microM for the neuronal, endothelial, and inducible nitric oxide synthase (NOS) isoforms, respectively. As measured at 100 microM l-arginine, neuronal NOS-catalyzed nitric oxide formation was inhibited 50% at a concentration of 25 microM C(3)-tris-malonyl-C(60)-fullerene. This inhibition was a multisite, positively cooperative inhibition with a Hill coefficient of 2.0. C(3)-tris-malonyl-C(60)-fullerene inhibited the arginine-independent NADPH-oxidase activity of nNOS with an IC(50) value of 22 microM but had no effects on its cytochrome c reductase activity at concentrations as high as 300 microM. The inhibition of nNOS activity by C(3)-tris-malonyl-C(60)-fullerene reduced the maximal velocity of product formation but did not alter the EC(50) value for activation by calmodulin. C(3)-tris-malonyl-C(60)-fullerene reduced the maximal velocity of citrulline formation by inducible NOS without altering the K(m) for l-arginine substrate or the EC(50) value for tetrahydrobiopterin cofactor. As measured by sucrose density gradient centrifugation, fully inhibitory concentrations of C(3)-tris-malonyl-C(60)-fullerene did not produce a dissociation of nNOS dimers into monomers. These observations are consistent with the proposal that C(3)-tris-malonyl-C(60)-fullerene inhibits the inter-subunit transfer of electrons, presumably by a reversible distortion of the dimer interface.

Publication types

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

MeSH terms

  • Animals
  • Carbon / chemistry*
  • Carbon / pharmacology*
  • Cattle
  • Centrifugation, Density Gradient
  • Citrulline / biosynthesis
  • Dose-Response Relationship, Drug
  • Endothelium, Vascular / enzymology
  • Escherichia coli / metabolism
  • Fullerenes*
  • Inhibitory Concentration 50
  • Kinetics
  • Macrophages / enzymology
  • Malonates / pharmacology
  • Mice
  • Models, Molecular
  • NADH Dehydrogenase / metabolism
  • NADPH Oxidases / metabolism
  • Neurons / enzymology
  • Nitric Oxide / biosynthesis
  • Nitric Oxide Synthase / antagonists & inhibitors*
  • Nitric Oxide Synthase / chemistry*
  • Nitric Oxide Synthase Type II
  • Pituitary Gland / enzymology
  • Protein Conformation
  • Protein Isoforms
  • Rats
  • Time Factors


  • Fullerenes
  • Malonates
  • Protein Isoforms
  • Citrulline
  • Nitric Oxide
  • Carbon
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type II
  • Nos2 protein, mouse
  • Nos2 protein, rat
  • NADPH Oxidases
  • NADH Dehydrogenase
  • fullerene C60