Tetrahydrobiopterin Binding to Macrophage Inducible Nitric Oxide Synthase: Heme Spin Shift and Dimer Stabilization by the Potent Pterin Antagonist 4-Amino-Tetrahydrobiopterin † Bernd Mayer,* ,‡ Chaoqun Wu, § Antonius C. F. Gorren, ‡ Silvia Pfeiffer, ‡ Kurt Schmidt, ‡ Pamela Clark, § Dennis J. Stuehr, § and Ernst R. Werner | Institut fu ¨ r Pharmakologie und Toxikologie, Karl-Franzens-UniVersita ¨ t Graz, UniVersita ¨ tsplatz 2, A-8010 Graz, Austria, Department of Immunology, CleVeland Clinic, 9500 Euclid AVenue, CleVeland, Ohio 44195, and Institut fu ¨ r Medizinische Chemie und Biochemie, UniVersita ¨ t Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria ReceiVed January 23, 1997; ReVised Manuscript ReceiVed March 31, 1997 X ABSTRACT: The characteristics of tetrahydrobiopterin (H 4 biopterin) binding to pteridine-free recombinant macrophage inducible nitric oxide synthase expressed in Escherichia coli were investigated with a special focus given to effects caused by 2,4-diamino-5,6,7,8-tetrahydro-6-(L-erythro-1,2-dihydroxypropyl)pteridine (4-amino-H 4 biopterin), a novel pterin-based inhibitor of nitric oxide synthase. The 4-amino compound completely inhibited enzyme stimulation by 10 μMH 4 biopterin with a half-maximally active concentration of 7.2 ( 0.39 μM, whereas H 2 biopterin and sepiapterin were much less potent. Binding studies using [ 3 H]H 4 biopterin at 4 °C revealed biphasic association of the radioligand according to two first-order reactions with apparent rate constants of 2.2 and 0.05 min -1 , each accounting for approximately 50% of total binding. Dissociation of [ 3 H]H 4 biopterin occurred with rate constants of 0.005 and 0.0028 min -1 in the absence and presence of L-arginine, respectively. Specific binding of 10 nM [ 3 H]H 4 biopterin was antagonized by unlabeled H 4 biopterin and its 4-amino analog with half-maximal effects at 84 ( 6 and 34 ( 3.2 nM, respectively. Binding of H 4 biopterin and 4-amino-H 4 biopterin was accompanied by a partial low spin to high spin conversion of the heme that was completed by L-arginine. Similarly, the active cofactor and the inhibitory 4-amino derivative both induced significant formation of stable protein dimers that survived during SDS electrophoresis, suggesting that the allosteric effects caused by H 4 biopterin do not explain sufficiently the essential role of the pteridine cofactor in NO biosynthesis. Nitric oxide is formed by enzymatic oxidation of the guanidino group of L-arginine by different nitric oxide synthases (NOS, 1 EC 1.14.13.39) (Griffith & Stuehr, 1995, Masters et al., 1996, Mayer, 1995). The neuronal (nNOS) and endothelial (eNOS) isoforms are constitutively expressed and require micromolar concentrations of free Ca 2+ for activity, whereas the isoform first described in murine macrophages (iNOS) is cytokine inducible and Ca 2+ - independent. Oxidation of L-arginine occurs via reductive activation of molecular oxygen catalyzed by a cytochrome P450-like heme iron localized in the oxygenase domain of the enzyme. The five electron oxidation of L-arginine is accompanied by an eight electron reduction of molecular oxygen with three exogenous electrons shuttled from the cofactor NADPH to heme by an FAD- and FMN-containing cytochrome P450 reductase domain. With this domain structure, NOS appears to be a self-sufficient cytochrome P450, resembling the soluble cytochrome P450 (BM-3) from Bacillus megaterium, which also contains oxygenase and reductase domains within a single polypeptide (Fulco, 1991). When activated by Ca 2+ /calmodulin in the presence of low concentrations of L-arginine or H 4 biopterin, nNOS exhibits NADPH oxidase activity resulting in formation of superoxide anions and H 2 O 2 due to uncoupling of oxygen reduction from L-arginine oxidation (Culcasi et al., 1994; Heinzel et al., 1992; Mayer et al., 1991; Pou et al., 1992). Uncoupling in the absence of L-arginine appears to be a specific feature of nNOS, since neither eNOS (List et al., 1997) nor iNOS (Abu- Soud & Stuehr, 1993; Olken & Marletta, 1993) exhibits considerable NADPH oxidase activity in the absence of a ligand bound to the substrate site. Unlike other P450s, NOS requires H 4 biopterin as a cofactor, but the precise function of the pteridine is not known (Mayer & Werner, 1995). Although H 4 biopterin may have a distinct function as a reactant in L-arginine oxidation, its allosteric effects resulting in profound changes in protein conformation are more obvious. Presence of the pteridine † This work was supported by Grants P 11478, P 10655, P 10859 (B.M.), P 10573 (K.S.), and P 11301 (E.R.W.) of the Fonds zur Fo ¨rderung der Wissenschaftlichen Forschung in O ¨ sterreich and by a National Institutes of Health Grant CA53914 (D.J.S.). D.J.S. is an Established Investigator of the American Heart Association. * To whom correspondence should be addressed: Institut fu ¨r Pharmakologie und Toxikologie, Karl-Franzens-Universita ¨t Graz, Uni- versita ¨tsplatz 2, A-8010 Graz, Austria. Tel: +43-316-380-5567. Fax: +43-316-380-9890. E-mail: mayer@kfunigraz.ac.at. ‡ Karl-Franzens-Universita ¨t Graz. § Cleveland Clinic. | Universita ¨t Innsbruck. X Abstract published in AdVance ACS Abstracts, July 1, 1997. 1 Abbreviations: NOS, nitric oxide synthase, eNOS, endothelial nitric oxide synthase (type III); iNOS, inducible nitric oxide synthase (type II); nNOS, neuronal nitric oxide synthase (type I); H4biopterin, 5,6,7,8- tetrahydro-L-erythro-biopterin ) 5,6,7,8-tetrahydro-6-(L-erythro-1,2- dihydroxypropyl)pterin; H2biopterin, 5,6,7,8-dihydro-L-erythro-biopterin ) 5,6,7,8-dihydro-6-(L-erythro-1,2-dihydroxypropyl)pterin; 4-amino- H4biopterin, 2,4-diamino-5,6,7,8-tetrahydro-6-(L-erythro-1,2-dihydroxy- propyl)pteridine; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]- 1-propanesulfonate; PAGE, polyacrylamide gel electrophoresis; EC50, concentration producing half-maximal effects; IC50, concentration producing half-maximal inhibition. 8422 Biochemistry 1997, 36, 8422 S0006-2960(97)00144-X CCC: $14.00 © 1997 American Chemical Society