〈Introduction〉Neuronal nitric oxide synthase (nNOS) is a Ca²⁺/calmodulin (CaM)-dependent enzyme that catalyzes the metabolism of L-arginine (Arg) to nitric oxide (NO) in the presence of NADPH and O₂. nNOS is only active as a dimer, however, a reduction of cofactor tetrahydrobiopterin (BH₄) levels drastically destabilizes the dimer and thereby uncoupling from NADPH and production of superoxide rather than NO occur. Protein polysulfidation at specific Cys residues plays pivotal roles in the protein function. In this study, we investigated the molecular mechanism of supersulfides-induced inactivation of nNOS.
〈Results and discussion〉Incubation of recombinant nNOS with Na₂S₄ in the presence of Arg and BH₄ resulted in a dose-dependent inhibition of NO production which was cancelled by reducing agent, dithiothreitol. Likewise, NADPH oxidation was inhibited with Na₂S₂ in the presence of BH₄ either with or without Arg but was not inhibited when both Arg and BH₄ were omitted. This indicates that inhibition targets NADPH oxidation with dimer formation of nNOS and without sign of uncoupling. The dimer/ monomer ratio of nNOS was not affected by Na₂S₄. Treatment of Na₂S₄ resulted in decreased ADP- but not CaM-agarose binding ability to nNOS. We successfully did generate the Na₂S₄-insensitive nNOS mutant in that its specific Cys reside within the NADPH-binding domain were mutated with serine. Thus, NADPH oxidation activity of dimerized nNOS is regulated by supersulfides via its regulatory cysteine residue modification.