Reactions Catalyzed by the Heme Domain of Inducible Nitric Oxide Synthase: Evidence for the Involvement of Tetrahydrobiopterin in Electron Transfer † Amy R. Hurshman ‡ and Michael A. Marletta* ,‡,§,| Howard Hughes Medical Institute, Department of Medicinal Chemistry, and Department of Biological Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109-0606 ReceiVed NoVember 1, 2001 ABSTRACT: The heme domain (iNOS heme ) of inducible nitric oxide synthase (iNOS) was expressed in Escherichia coli and purified to homogeneity. Characterization of the expressed iNOS heme shows it to behave in all respects like full-length iNOS. iNOS heme is isolated without bound pterin but can be readily reconstituted with (6R)-5,6,7,8-tetrahydro-L-biopterin (H 4 B) or other pterins. The reactivity of pterin- bound and pterin-free iNOS heme was examined, using sodium dithionite as the reductant. H 4 B-bound iNOS heme catalyzes both steps of the NOS reaction, hydroxylating arginine to N G -hydroxy-L-arginine (NHA) and oxidizing NHA to citrulline and •NO. Maximal product formation (0.93 ( 0.12 equiv of NHA from arginine and 0.83 ( 0.08 equiv of citrulline from NHA) requires the addition of 2 to 2.5 electron equiv. Full reduction of H 4 B-bound iNOS heme with dithionite also requires 2 to 2.5 electron equiv. These data together demonstrate that fully reduced H 4 B-bound iNOS heme is able to catalyze the formation of 1 equiv of product in the absence of electrons from dithionite. Arginine hydroxylation requires the presence of a bound, redox-active tetrahydropterin; pterin-free iNOS heme or iNOS heme reconstituted with a redox-inactive analogue, 6(R,S)-methyl-5-deaza-5,6,7,8-tetrahydropterin, did not form NHA under these conditions. H 4 B has an integral role in NHA oxidation as well. Pterin-free iNOS heme oxidizes NHA to citrulline, N δ - cyanoornithine, an unidentified amino acid, and NO - . Maximal product formation (0.75 ( 0.01 equiv of amino acid products) requires the addition of 2 to 2.5 electron equiv, but reduction of pterin-free iNOS heme requires only 1 to 1.5 electron equiv, indicating that both electrons for the oxidation of NHA by pterin- free iNOS heme are derived from dithionite. These data provide strong evidence that H 4 B is involved in electron transfer in NOS catalysis. The biosynthesis of •NO is catalyzed by the enzyme nitric oxide synthase (NOS, 1 EC 1.14.13.39) in a reaction that also forms citrulline from the amino acid L-arginine (for reviews, see refs 1-3). In this five-electron oxidation, •NO is derived from one of the terminal guanidino nitrogens of arginine. The reaction proceeds in two steps, both of which require NADPH and O 2 as cosubstrates. The first step of the reaction is the hydroxylation of arginine, forming N G -hydroxy-L- arginine (NHA) as an intermediate (4-6). NHA is further oxidized by three electrons in the second step to form citrulline and •NO. Three isoforms of NOS have been characterized: a particulate, constitutive enzyme from vas- cular endothelium (eNOS), a soluble, constitutive enzyme from neuronal cells (nNOS), and an inducible enzyme, best characterized from murine macrophages (iNOS) (7). All of the isoforms are homodimeric and bind an equivalent each of FAD, FMN (8-10), and protoporphyrin IX heme (11- 13) per subunit. Full activity also requires one bound H 4 B per monomer (10, 14, 15). The roles of the enzyme-bound heme and H 4 B in the reaction mechanism are not fully understood. CO inhibition studies have suggested a catalytic role for the heme in both steps of the NOS reaction (16, 11). Further evidence for the involvement of the heme in NHA oxidation comes from NOS reactions where hydrogen peroxide is substituted for NADPH and O 2 (peroxide-shunt reactions). The products of the peroxide-shunt reactions are consistent with a heme ferric- peroxide nucleophile as an intermediate in the NADPH- dependent oxidation of NHA (17, 18). A model for the enzymatic oxidation of NHA to citrulline and •NO, using an Fe III porphyrin and O 2 , has also been reported (19). The role of the heme in the hydroxylation of arginine has been less clear. Crystal structures of several NOS heme domains † This research was supported by Howard Hughes Medical Institute and NIH Grant CA 50414. A.R.H. was supported by NIH Grant T32- GM07767, a Regents’ Fellowship from the University of Michigan, and an American Foundation for Pharmaceutical Education Fellowship. * To whom correspondence should be addressed at University of California, Berkeley, Department of Chemistry, 211 Lewis Hall, Berkeley, CA 94720-1460. Phone: (510) 643-9325; Fax: (510) 643- 9388; E-mail: marletta@cchem.berkeley.edu. ‡ Department of Medicinal Chemistry, University of Michigan. § Howard Hughes Medical Institute, University of Michigan. | Department of Biological Chemistry, University of Michigan. 1 Abbreviations: NOS, nitric oxide synthase; eNOS, endothelial NOS; nNOS, neuronal NOS; iNOS, inducible NOS; iNOSheme, heme domain of inducible NOS; NHA, N G -hydroxy-L-arginine; H4B, (6R)- 5,6,7,8-tetrahydro-L-biopterin; 7,8-H2B, (6R)-7,8-dihydro-L-biopterin; MPH4, 6(R,S)-methyl-5,6,7,8-tetrahydropterin; DZPH4, 6(R,S)-methyl- 5-deaza-5,6,7,8-tetrahydropterin; DTT, dithiothreitol; CN-orn, N δ - cyanoornithine; HEPES, 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid; Tris-HCl, Tris[hydroxymethyl]aminomethane hydrochloride; IPTG, isopropyl--D-thiogalactopyranoside; PVDF, polyvinylidine fluoride; PCA, protocatechuic acid; PCD, protocatechuate 3,4-dioxygenase; Ni- NTA, nickel-nitrilotriacetic acid agarose; BSA, bovine serum albumin; NDA, 2,3-naphthalene dicarboxaldehyde; EDTA, ethylenediamine- N,N,N′,N′-tetraacetic acid; TFA, trifluoroacetic acid; EPR, electron paramagnetic resonance spectroscopy; PCR, polymerase chain reaction; MALDI, matrix-assisted laser desorption/ionization; HPLC, high- performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; ICP/MS, inductively coupled plasma/mass spectrometry. 3439 Biochemistry 2002, 41, 3439-3456 10.1021/bi012002h CCC: $22.00 © 2002 American Chemical Society Published on Web 02/12/2002