Nitric oxide synthases (NOSs), which catalyze the formation of the ubiquitous biological messenger molecule nitric oxide, represent unique cytochrome P‐450s, containing reductase and mono‐oxygenase domains within one polypeptide and requiring tetrahydrobiopterin as cofactor. To investigate whether tetrahydrobiopterin functions as an allosteric effector of NOS, we have analyzed the effect of the pteridine on the conformation of neuronal NOS purified from porcine brain by means of circular 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 neuronal NOS was neither modulated nor stabilized by tetrahydrobiopterin, and the pteridine did not affect the quaternary structure of the protein, which appears to be an elongated homodimer with an axial ratio of approximately 20/1 under native conditions. Low temperature SDS‐polyacrylamide gel electrophoresis revealed that tetrahydrobiopterin and L‐arginine synergistically convert neuronal NOS into an exceptionally stable, non‐covalently linked homodimer surviving in 2% SDS and 5% 2‐mercaptoethanol. Ligand‐induced formation of an SDS‐resistant dimer is unprecedented and suggests a novel role for tetrahydrobiopterin and L‐arginine in the allosteric regulation of protein subunit interactions.

Neuronal nitric-oxide (NO) synthase contains FAD, FMN, heme, and tetrahydrobiopterin as prosthetic groups and represents a multifunctional oxidoreductase catalyzing oxidation of L-arginine to L-citrulline and NO, reduction of molecular oxygen to superoxide, and electron transfer to cytochromes. To investigate how binding of the prosthetic heme moiety is related to enzyme activities, cofactor, and L-arginine binding, as well as to secondary and quaternary protein structure, we have purified and characterized heme-deficient neuronal NO synthase. The heme-deficient enzyme, which had preserved its cytochrome c reductase activity, contained FAD and FMN, but virtually no tetrahydrobiopterin, and exhibited only marginal NO synthase activity. By means of gel filtration and static light scattering, we demonstrate that the heme-deficient enzyme is a monomer and provide evidence that heme is the sole prosthetic group controlling the quaternary structure of neuronal NO synthase. CD spectroscopy showed that most of the structural elements found in the dimeric holoenzyme were conserved in heme-deficient monomeric NO synthase. However, in spite of being properly folded, the heme-deficient enzyme did bind neither tetrahydrobiopterin nor the substrate analog N(G)-nitro-L-arginine. Our results demonstrate that the prosthetic heme group of neuronal NO synthase is requisite for dimerization of enzyme subunits and for the binding of amino acid substrate and tetrahydrobiopterin.

Depolarized dynamic light scattering (DDLS) experiments are reported on three different systems:  on tobacco mosaic virus as a well investigated sample; on PEO−PPO−PEO triblock copolymer micelles, which show sphere-to-rod transition with increasing temperature; and on a microemulsion of the type water/octane/C i E j . DDLS measurements were performed using highly discriminating polarizers and single-mode fiber detection at different scattering angles, obtaining decay rates Γ VH versus scattering angle. The rotational and translational diffusion coefficients available from these plots were taken to evaluate size parameters of the systems using Broersma's expressions for a stiff rod. Good agreement with theory and literature was found in the case of tobacco mosaic virus and at concentrations below the overlap concentration c* for the other two systems.

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