Tetrahydrobiopterin-free neuronal nitric oxide synthase: evidence for two identical highly anticooperative pteridine binding sites.
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.
Gorren
A C
AC
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Austria.
List
B M
BM
Schrammel
A
A
Pitters
E
E
Hemmens
B
B
Werner
E R
ER
Schmidt
K
K
Mayer
B
B
D000818:Animals; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives / Q000032:analysis; D019281:Dimerization; D006418:Heme / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D015394:Molecular Structure; D009474:Neurons / Q000201:enzymology; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011621:Pteridines / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry
D011621:Pteridines; D011994:Recombinant Proteins; D001708:Biopterins; D006418:Heme; D019001:Nitric Oxide Synthase; C003402:sapropterin