C₆₀-Fullerene Monomalonate Adducts Selectively Inactivate Neuronal Nitric Oxide Synthase by Uncoupling the Formation of Reactive Oxygen Intermediates from Nitric Oxide Production

Abstract

C(60)-Fullerene monomalonate adducts inactivate selectively the neuronal nitric oxide synthase isoform in a manner completely preventable by the concurrent presence of superoxide dismutase and catalase. This inactivation is time-, fullerene concentration-, and turnover-dependent and is not reversible by dilution. The di(carboxypropan-3-ol)methano-[60]-fullerene (diol adduct) has no effect on NADPH consumption by nNOS as measured in the absence of arginine substrate, but dramatically increases NADPH consumption in the presence of arginine. This fullerene-enhanced NADPH consumption is linked to oxygen as electron acceptor and is accompanied by the increased production of hydrogen peroxide. These effects of fullerene monomalonate adducts are unique to the nNOS isoform and are not observed using either the iNOS or the eNOS isoform. The inhibitory effects of fullerene monomalonate adducts are unaltered and insurmountable by increased concentrations of arginine, tetrahydrobiopterin, or calmodulin. These observations indicate that fullerene monomalonate adducts uncouple in the presence of arginine the formation of reactive oxygen intermediates from NO production by nNOS. These reactive oxygen intermediates dissociate from the enzyme and, acting from solution, inactivate NOS NO forming activity.