EPR and ENDOR Characterization of Intermediates in the Cryoreduced Oxy-Nitric Oxide Synthase Heme Domain with Bound L-Arginine or N G -Hydroxyarginine ² Roman Davydov, ‡ Amy Ledbetter-Rogers, § Pavel Marta ´sek, |,⊥ Mikhail Larukhin, ‡ Masanori Sono, # John H. Dawson,* ,# Bettie Sue Siler Masters,* ,| and Brian M. Hoffman* ,‡ Department of Chemistry, Northwestern UniVersity, EVanston, Illinois 60201, Department of Chemistry and Biochemistry, College of Charleston, Charleston, South Carolina 29424, Department of Chemistry and Biochemistry, UniVersity of South Carolina, Columbia, South Carolina 29208, Department of Biochemistry, The UniVersity of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900, and Department of Pediatrics, Center of IntegratiVe Genomics, 1 st School of Medicine, Charles UniVersity, Prague, Czech Republic ReceiVed May 2, 2002; ReVised Manuscript ReceiVed June 25, 2002 ABSTRACT: Reconstitution of the endothelial nitric oxide synthase heme domain (NOS) with the catalytically noncompetent 4-aminotetrahydrobiopterin has allowed us to prepare at -40 °C the oxyferrous-NOS- substrate complexes of both L-arginine (Arg) and N G -hydroxyarginine (NOHA). We have radiolytically cryoreduced these complexes at 77 K and used EPR and ENDOR spectroscopies to characterize the initial products of reduction, as well as intermediates that arise during stepwise annealing to higher temperatures. Peroxo-ferri-NOS is the primary product of 77 K cryoreduction when either Arg or NOHA is the substrate. Proton ENDOR spectra of this state suggest that the peroxo group is H-bonded to a [guanidinium-water] network that forms because the binding of O 2 to the ferroheme of NOS recruits H 2 O. At no stage of reaction/annealing does one observe an EPR signal from a hydroperoxo-ferri state with either substrate. Instead, peroxo-ferri-NOS-substrate complexes convert to a product-state intermediate at the extremely low temperature of 165-170 K. EPR and proton ENDOR spectra of the intermediate formed with Arg as substrate support the suggestion that the reaction involves the formation and attack of Compound I. Within the time/temperature resolution of the present experiments, samples with Arg and NOHA as substrate behave the same in the initial steps of cryoreduction/annealing, despite the different acid/base characteristics of the two substrates. This leads us to discuss the possibility that ambient-temperature catalytic conversion of both substrates is initiated by reduction of the oxy-ferroheme to the hydroperoxo-ferriheme through a coupled proton-electron transfer from a heme-pocket reductant, and that Arg may provide the stoichiometrically second proton of catalysis. Nitric oxide synthase (NOS) 1 catalyzes the formation of NO from L-arginine (Arg) in two dioxygen-activating stages, eq 1, the first of which produces N G -hydroxyarginine (NOHA) and the second of which generates NO and citrulline (1-6). Each stage begins with the reduction of ferri- to ferro-NOS, which binds dioxygen. The first stage has the stoichiometry of a ‘classical’ mixed-function hydroxylation, requiring a second electron and two protons to generate NOHA and water. The holoenzyme employs NADPH as the ultimate source of electrons, but dithionite also can act as an electron source to the NOS heme domain (7). Rapid-kinetic studies have provided evidence that tetrahydrobiopterin (H 4 B), an essential cofactor bound in the heme pocket, then provides the ‘second’ electron, reducing the oxyferrous heme and generating a pterin radical (8-10), which must be rereduced prior to the next cycle. The reaction of NOHA to form NO and citrulline, the second stage of eq 1, does not stoichio- metrically require a second electron, but nonetheless is thought to involve reduction of the oxyferrous heme, perhaps by the pterin as in step one, or else by NOHA itself (7, 11). It is widely thought that the reduction of the oxyferrous- NOS-Arg generates the peroxo-ferri-heme, which then proceeds through hydroperoxo-ferri-NOS to Compound I (Scheme 1), which is the actual hydroxylating intermediate. Precisely this scheme indeed applies to P450cam hydroxyl- ations, for which it was first proposed (12-15), but it is not universal: the hydroxylating intermediate of heme oxygenase (HO) is the hydroperoxo-ferri form, not Compound I (16, 17), and the peroxo-ferri form appears to be involved in a nitrile-to-amide conversion by P450 3A4 (18). In the ² We acknowledge grants from the National Institutes of Health [B.M.H. (HL13531), J.H.D. (GM26730), and B.S.S.M. (GM52419)], The Robert A. Welch Foundation [B.S.S.M. (AQ-1192)], and MSMT, Czech Republic [P.M. (LN 0VA079)]. ‡ Northwestern University. § College of Charleston. | The University of Texas Health Science Center at San Antonio. ⊥ Charles University. # University of South Carolina. 1 Abbreviations: NOS, endothelial nitric oxide synthase heme domain; Arg, L-arginine; NOHA, N G -hydroxyarginine; H4B, tetrahydro- biopterin; ENDOR, electron nuclear double resonance; EPR, electron paramagnetic resonance; HO, heme oxygenase; P450cam, cytochrome P450cam; NADPH, reduced nicotine adenine dinucleotide phosphate; EDTA, ethylenediaminetetraacetate. Arg 9 8 O 2 , 2e - , 2H + NOS NOHA 9 8 O 2 , 1e - , 1H + NOS citrulline + NO • (1) BATCH: bi8d25 USER: jdm69 DIV: @xyv04/data1/CLS_pj/GRP_bi/JOB_i33/DIV_bi0260637 DATE: July 30, 2002 10375 Biochemistry 2002, 41, 10375-10381 10.1021/bi0260637 CCC: $22.00 © 2002 American Chemical Society Published on Web 07/25/2002