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

C-3-tris-malonyl-C-60-fullerene and D-3-tris-malonyl-C-60-fullerene derivat ives inhibit citrulline and NO formation by all three nitric oxide synthase isoforms in a manner fully reversible by dilution. The inhibition of citru lline formation by C-3-tris-malonyl-C-60-fullerene occurs with IC50 values of 24, 17, and 123 mu M for the neuronal, endothelial, and inducible nitric oxide synthase (NOS) isoforms, respectively. As measured at 100 mu M L-arg inine, neuronal NOS-catalyzed nitric oxide formation was inhibited 50% at a concentration of 25 mu M 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 IC50 value of 22 mu M but had no eff ects on its cytochrome c reductase activity at concentrations as high as 30 0 mu M. 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 EC5 0 value for activation by calmodulin, C-3-tris-malonyl-C-60-fullerene reduc ed the maximal velocity of citrulline formation by inducible NOS without al tering the K-m for L-arginine substrate or the EC50 value for tetrahydrobio pterin cofactor. As measured by sucrose density gradient centrifugation, fu lly inhibitory concentrations of C-3-tris-malonyl-C-60-fullerene did not pr oduce a dissociation of nNOS dimers into monomers, These observations are c onsistent with the proposal that C-3-tris-malonyl-C-60-fuIlerene inhibits t he inter-subunit transfer of electrons, presumably by a reversible distorti on of the dimer interface. (C) 2000 Academic Press.