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Recent years have seen a massive growth of interest in the biological effects of nitric oxide (NO): It acts as a signaling molecule in blood vessels and the brain, and as a defense against pathogens in the immune system. Discussed are the chemical events underlying the physiology of this versatile little molecule.

The properties of neuronal nitric oxide synthase containing one tetrahydrobiopterin (BH4) per dimer [nNOS(BH4+)] were compared to those of the BH4-free enzyme [nNOS(BH4-)]. The stimulation by BH4 of the formation of L-citrulline at the expense of H2O2 production unambiguously demonstrated that BH4 is essential in coupling reductive oxygen activation to Arg oxidation. The clear difference between the Stokes radii of nNOS(BH4-) and nNOS(BH4+) indicates that the introduction of one BH4 per dimer significantly changes the enzyme structure. Whereas the heme in nNOS(BH4+) was primarily high-spin, nNOS(BH4-) contained mainly low-spin heme. This was slowly converted into the high-spin form with Arg and/or BH4, with a rate that was independent of the concentration of either compound. Dithiothreitol inhibited the Arg/BH4-induced spin conversion by stabilizing low-spin heme. Formation of high-spin heme, with rates varying from 0.04 to 0.4 min-1, always correlated to an equally fast increase in activity. Radioligand binding studies showed the rapid association (within 20 s) of BH4 to nNOS(BH4-), but not to nNOS(BH4+), after preincubation with Arg. Complete and monophasic dissociation of radioligand occurred in the presence of excess unlabeled BH4, demonstrating the exchangeability of high-affinity bound BH4. Studies of the association of NG-nitro-L-arginine (L-NNA) to nNOS(BH4+) revealed that excess BH4 increased the amount of bound L-NNA 2-fold. Most of the binding data are explained by a model in which nNOS dimers accommodate two identical BH4- and Arg/L-NNA-binding sites, with cooperativity between Arg- and BH4-binding and anticooperativity between the BH4-binding sites.

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