title: Characterization of heme-deficient neuronal nitric-oxide synthase reveals a role for heme in subunit dimerization and binding of the amino acid substrate and tetrahydrobiopterin.
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.
authors.affiliation: Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Austria.
authors.forename: P
authors.initials: P
authors.lastname: Klatt
authors.forename: S
authors.initials: S
authors.lastname: Pfeiffer
authors.forename: B M
authors.initials: BM
authors.lastname: List
authors.forename: D
authors.initials: D
authors.lastname: Lehner
authors.forename: O
authors.initials: O
authors.lastname: Glatter
authors.forename: H P
authors.initials: HP
authors.lastname: Bächinger
authors.forename: E R
authors.initials: ER
authors.lastname: Werner
authors.forename: K
authors.initials: K
authors.lastname: Schmidt
authors.forename: B
authors.initials: B
authors.lastname: Mayer
mesh_terms: D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D002956:Citrulline / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008027:Light; D046911:Macromolecular Substances; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019335:Nitroarginine; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051381:Rats; D012542:Scattering, Radiation; D013816:Thermodynamics
chemical_list: D007527:Isoenzymes; D046911:Macromolecular Substances; D005182:Flavin-Adenine Dinucleotide; D019335:Nitroarginine; D001708:Biopterins; D002956:Citrulline; D006418:Heme; D005486:Flavin Mononucleotide; D001120:Arginine; D019001:Nitric Oxide Synthase; C003402:sapropterin