Inhibition of nitric oxide synthase activity by Zn2+ ion.
We have found neural nitric oxide synthase (nNOS) activity to be completely and reversibly inhibited by Zn2+ ion with an apparent Ki of 30 microM. 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 approximately 430 nm and a trough at approximately 395 nm, with an apparent spectral binding constant of 50 microM. These spectral changes are consistent with a Zn(2+)-dependent change in the spin-state equilibrium of the heme iron in nNOS. The spectral binding constant for L-arginine binding to nNOS (approximately 1.5 microM) is not significantly affected by the presence of 50 microM Zn2+, indicating that Zn(2+)-dependent inhibition of nNOS activity is not due to interference with substrate binding. The estimated maximal change in nNOS absorbance at approximately 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 50 microM Zn2+, which reflects the increased initial amount of low-spin ferric heme iron present. These data indicate that Zn(2+)-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.
Persechini
A
A
Department of Physiology, University of Rochester Medical Center, New York 14642, USA.
McMillan
K
K
Masters
B S
BS
D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D002413:Cations, Divalent; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007668:Kidney; D009249:NADP / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D051381:Rats; D011994:Recombinant Proteins; D013053:Spectrophotometry; D015032:Zinc / Q000494:pharmacology*
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D009249:NADP; D019001:Nitric Oxide Synthase; D015032:Zinc; D002122:Calcium Chloride