We have found neural nitric oxide synthase (nNOS) activity to be completely and reversibly inhibited by Zn2+ ion with an apparent Ki of 30µM. Zn2+ blocks NADPH-dependent reduction of heme iron in nNOS and also blocks the calmodulin-dependent superoxide-mediated cytochrome c reductase 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 characterized by the appearance of a peak at ~430 nm and a trough at ~395 nm, with an apparent spectral binding constant of 50 µM. These spectral changes are consistent with a Zn2+-dependent change in the spin-state equilibrium of the heme iron in nNOS. The spectral binding constant for L-arginine binding to nNOS (~ 1.5 µm) is not significantly affected by the presence of 50µM Zn2+, indicating that Zn2+-dependent inhibition of nNOS activity is not due to interference with substrate binding. The estimated maximal change in nNOS absorbance at ~418 nm caused by the L-arginine-dependent conversion of the ferric heme iron from hexacoordinate low-spin to pentacoordinate high-spin is increased by 50% in the presence of 50µM Zn2+, which reflects the increased initial amount of low-spin ferric heme iron present. These data indicate that Zn2+-dependent inhibition 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.
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