Abstract
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.
Highlights
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
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
To investigate the role of heme in neuronal NOS (nNOS) structure and function, we attempted to purify recombinant heme-deficient nNOS from a baculovirus overexpression system
Summary
Materials—L-[2,3,4,5-3H]Arginine hydrochloride (57 Ci/mmol) and NG-nitro-L-[2,3,4,5-3H]arginine hydrochloride (56 Ci/mmol) were purchased from MedPro (Amersham), Vienna, Austria. Incubations were performed for 10 min at 37 °C in 0.1 ml of 50 mM triethanolamine/HCl buffer, pH 7.0, containing 0.6 –2.4 pmol of nNOS, 0.1 mM L-[2,3,4,5-3H]arginine (ϳ80,000 cpm), 0.5 mM CaCl2, 10 g/ml calmodulin, 0.2 mM NADPH, 10 M H4biopterin, 5 M FMN, and 5 M FAD. NNOS (18 – 40 pmol) was incubated for 10 min at 37 °C with 12 nM L-[3H]NNA (ϳ70 nCi) or [3H]H4biopterin (ϳ17 nCi) and increasing concentrations of the respective unlabeled ligand (10 nM-10 M) in 0.1 ml of a 50 mM triethanolamine/HCl buffer, pH 7.0. NNOS (ϳ150 pmol) was incubated for 5 min at 37 °C in 50 l of 50 mM triethanolamine/HCl buffer (pH 7.0) in the absence or presence of H4biopterin (0.1 mM) and L-arginine (1 mM). Calculations of ⌬⑀ values were based on amino acid analysis, and the secondary structure of nNOS was computed by means of the variable selection method using a set of 33 reference proteins [46]
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