STRUCTURAL-ANALYSIS OF PORCINE BRAIN NITRIC-OXIDE SYNTHASE REVEALS A ROLE FOR TETRAHYDROBIOPTERIN AND L-ARGININE IN THE FORMATION OF AN SDS-RESISTANT DIMER

Nitric oxide synthases (NOSs), which catalyze the formation of the ubi quitous biological messenger molecule nitric oxide, represent unique c ytochrome P-450s, containing reductase and mono-oxygenase domains with in one polypeptide and requiring tetrahydrobiopterin as cofactor. To i nvestigate whether tetrahydrobiopterin functions as an allosteric effe ctor of NOS, we have analyzed the effect of the pteridine on the confo rmation of neuronal NOS purified from porcine brain by means of circul ar dichroism, velocity sedimentation, dynamic light scattering and SDS -polyacrylamide gel electrophoresis. We report for the first time the secondary structure of NOS, showing that the neuronal isozyme contains 30% alpha-helix, 14% antiparallel beta-sheet, 7% parallel beta-sheet, 19% turns and 31% other structures. The secondary structure of neuron al NOS was neither modulated nor stabilized by tetrahydrobiopterin, an d the pteridine did not affect the quaternary structure of the protein , which appears to be an elongated homodimer with an axial ratio of si milar to 20/1 under native conditions. Low temperature SDS-polyacrylam ide gel electrophoresis revealed that tetrahydrobiopterin and L-argini ne synergistically convert neuronal NOS into an exceptionally stable, noncovalently linked homodimer surviving in 2% SDS and 5% 2-mercaptoet hanol. Ligand-induced formation of an SDS-resistant dimer is unprecede nted and suggests a novel role for tetrahydrobiopterin and L-arginine in the allosteric regulation of protein subunit interactions.