TETRAHYDROBIOPTERIN-FREE NEURONAL NITRIC-OXIDE SYNTHASE - EVIDENCE FOR 2 IDENTICAL HIGHLY ANTICOOPERATIVE PTERIDINE BINDING-SITES

The properties of neuronal nitric oxide synthase containing one tetrah ydrobiopterin (BH4) per dimer [nNOS(BH4+)] were compared to those of t he BH4-free enzyme [nNOS(BH4-)]. The stimulation by BH4 of the formati on of L-citrulline at the expense of H2O2 production unambiguously dem onstrated that BH4 is essential in coupling reductive oxygen activatio n to Arg oxidation. The clear difference between the Stokes radii of n NOS(BH4-) and nNOS(BH4+) indicates that the introduction of one BH4 pe r dimer significantly changes the enzyme structure. Whereas the heme i n 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 e ither compound. Dithiothreitol inhibited the Arg/BH4-induced spin conv ersion 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 equal ly fast increase in activity. Radioligand binding studies showed the r apid association (within 20 s) of BH4 to nNOS(BH4-), but not to nNOS(B H4+), after preincubation with Arg. Complete and monophasic dissociati on of radioligand occurred in the presence of excess unlabeled BH4, de monstrating the exchangeability of high-affinity bound BI-4. Studies o f the association of NG-nitro-L-arginine (L-NNA) to nNOS(BH4-) reveale d 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 accommo date two identical BH4- and Arg/L-NNA-binding sites, with cooperativit y between Arg- and BH4-binding and anticooperativity between the BH4-b inding sites.