Inhibition of Nitric Oxide Synthase Isoforms by Tris-Malonyl-C60-Fullerene Adducts

C3-tris-malonyl-C60-fullerene and D3-tris-malonyl-C60-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 C3-tris-malonyl-C60-fullerene occurs with IC50 values of 24, 17, and 123 μM for the neuronal, endothelial, and inducible nitric oxide synthase (NOS) isoforms, respectively. As measured at 100 μM l-arginine, neuronal NOS-catalyzed nitric oxide formation was inhibited 50% at a concentration of 25 μM C3-tris-malonyl-C60-fullerene. This inhibition was a multisite, positively cooperative inhibition with a Hill coefficient of 2.0. C3-tris-malonyl-C60-fullerene inhibited the arginine-independent NADPH-oxidase activity of nNOS with an IC50 value of 22 μM but had no effects on its cytochrome c reductase activity at concentrations as high as 300 μM. The inhibition of nNOS activity by C3-tris-malonyl-C60-fullerene reduced the maximal velocity of product formation but did not alter the EC50 value for activation by calmodulin. C3-tris-malonyl-C60-fullerene reduced the maximal velocity of citrulline formation by inducible NOS without altering the Km for l-arginine substrate or the EC50 value for tetrahydrobiopterin cofactor. As measured by sucrose density gradient centrifugation, fully inhibitory concentrations of C3-tris-malonyl-C60-fullerene did not produce a dissociation of nNOS dimers into monomers. These observations are consistent with the proposal that C3-tris-malonyl-C60-fullerene inhibits the inter-subunit transfer of electrons, presumably by a reversible distortion of the dimer interface.