INHIBITION OF NITRIC-OXIDE SYNTHASE ACTIVITY BY ZN2+ ION

We have found neural nitric oxide synthase (nNOS) activity to be compl etely and reversibly inhibited by Zn2+ ion with an apparent K-i of 30 mu M. Zn2+ blocks NADPH-dependent reduction of heme iron in nNOS and a lso blocks the calmodulin-dependent superoxide-mediated cytochrome c r eductase activity exhibited by nNOS. However, Zn2+ ion has no apparent effect on the calmodulin-independent direct reduction of cytochrome c by nNOS. Zn2+ ion induces perturbation difference spectra in nNOS cha racterized by the appearance of a peak at similar to 430 nm and a trou gh at similar to 395 nm, with an apparent spectral binding constant of 50 mu M. These spectral changes are consistent with a Zn2+-dependent change in the spin-state equilibrium of the heme iron in nNOS. The Spe ctral binding constant for L-arginine binding to nNOS (similar to 1.5 mu M) is not significantly affected by the presence of 50 mu M Zn2+, i ndicating that Zn2+-dependent inhibition of nNOS activity is not due t o interference with substrate binding. The estimated maximal change in nNOS absorbance at similar to 418 nm caused by the L-arginine-depende nt conversion of the ferric heme iron from hexacoordinate low-spin to pentacoordinate high-spin is increased by 50% in the presence of 50 mu M Zn2+, which reflects the increased initial amount of low-spin ferri c heme iron present. These data indicate that Zn2+-dependent inhibitio n of nNOS activity is due to binding of Zn2+ to the hemoprotein domain in the enzyme and that inhibition is associated with perturbations in the environment of the heme iron that appear to block its ability to mediate oxygen reduction.