Critical Review Tetrahydrobiopterin in Nitric Oxide Synthase

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1 Critical Review Tetrahydrobiopterin in Nitric Oxide Synthase Jesus Tejero 1 Dennis Stuehr 2,* 1 Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA 2 Department of Pathobiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA Abstract Summary: Nitric oxide synthase (NOS) is a critical enzyme for the production of the messenger molecule nitric oxide (NO) from L-arginine. NOS enzymes require tetrahydrobiopterin as a cofactor for NO synthesis. Besides being one of the few enzymes to use this cofactor, the role of tetrahydrobiopterin in NOS catalytic mechanism is different from other enzymes: during the catalytic cycle of NOS, tetrahydrobiopterin forms a radical species that is again reduced, thus effectively regenerating after each NO synthesis cycle. In this review, we summarize our current knowledge about the role of tetrahydrobiopterin in the structure, function, and catalytic mechanism of NOS enzymes. VC 2013 IUBMB Life, 65(4): , 2013 Keywords: nitric oxide synthase; nitric oxide; pterin radicals; tetrahydrobiopterin Introduction Nitric oxide synthases (NOS, EC ) are enzymes that catalyze the formation of nitric oxide (NO) from L-arginine. NO is an important signaling molecule that participates in a number of biological processes, including neurotransmission, vasodilation, and immune response (1). NOS proteins are active as homodimers, and each monomer consists of two well-defined domains. The N-terminal oxygenase domain binds heme, L-arginine, and (6R-)5,6,7,8-tetrahydrobiopterin (H 4 B). The C-terminal reductase domain binds flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide phosphate (NADPH) and provides electrons to the oxygenase domain from NADPH. Both domains can be VC 2013 International Union of Biochemistry and Molecular Biology, Inc. Volume 65, Number 4, April 2013, Pages Abbreviations: CaM, calmodulin; enos, endothelial nitric oxide synthase; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; H 2 B, 7,8- dihydrobiopterin; H 4 B, (6R-)5,6,7,8-tetrahydrobiopterin; H 4 F, tetrahydrofolate; inos, inducible nitric oxide synthase; NADPH, nicotinamide adenine dinucleotide phosphate; nnos, neuronal nitric oxide synthase; NOHA, N x - hydroxy arginine; NOS, nitric oxide synthase *Address for correspondence to: Dennis J. Stuehr, Department of Pathobiology (NC-22), The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA. Tel: Fax: stuehrd@ccf.org. Received 7 December 2012; accepted 25 December 2012 DOI: /iub.1136 Published online 26 February 2013 in Wiley Online Library (wileyonlinelibrary.com) expressed and purified separately, and conserve their ligand binding and reactivity. Between the oxygenase and reductase domains, a short sequence (30 40 aminoacids) provides a binding site for calmodulin (CaM) (Fig. 1). The oxygenase domain of NOS catalyzes the oxidation of L- arginine to L-citrulline and NO in two sequential oxidation steps (Scheme 1). In the first step, L-arginine is hydroxylated to make N x -hydroxy-l-arginine (NOHA) in a process that requires one molecule of NADPH and one molecule of oxygen per mol of L-arginine reacted. In the second step, NOHA is oxidized to L-citrulline and NO and 1 molecule of oxygen and 0.5 molecules of NADPH are required. The three mammalian isoforms of NOS show high sequence homology but greatly differ in their localization and regulation. The inducible isoform (inos) has a high affinity for CaM and is constitutively active; this isoform is mainly regulated at the transcriptional level. Endothelial (enos) and neuronal (nnos) enzymes are constitutively expressed and are reversibly activated by calcium through the binding of Ca 2þ -containing CaM. Related proteins have been discovered in bacteria but consisting of only the oxygenase domain (2). The bacterial NOS enzymes also contain heme but are less stringent in their pterin requirements and can often bind tetrahydrofolate (H 4 F) and H 4 B with similar affinity. Due to the absence of a connected reductase domain, they must receive electrons from other electron transfer proteins (2 4). The recently discovered NOS from Sorangium cellulosum is a notable exception to this rule (5). The general biochemistry of NOS enzymes has been extensively reviewed from the biophysical (6 9) and clinical 358 IUBMB Life

2 cycle involving H 4 BandH 2 B, as in aromatic amino acid hydroxylases was considered, but this would require one H 4 Bmolecule per cycle, in contradiction with experimental data (15,17). Strong evidence indicated that H 4 B enhanced dimer formation (18,19). However, the evidences of some redox effect did slowly accumulate. Single turnover experiments indicated that the stability of the heme-oxy complex was dependent on H 4 Bpresence, and H 4 B was required for the reaction to advance past the formation of a ferrous dioxygen/ferric superoxide complex (20). Other results indicated that H 4 B analogs were able to catalyze L- arginine hydroxylation to a variable extent, but all were reduced forms (21). It was even unclear if H 4 B was required for the NOHA oxidation step (22). The detection of a H 4 B radical (23 26) and the coupling of this radical formation with product formation (26) finally lead to the currently accepted notion the decay of the ferrous dioxygen/ferric superoxide complex is linked to the formation of a H 4 B radical species (8,9,27,28). FIG 1 Schematic mechanism of electron transfer in NOS enzymes. In the presence of H 4 B, the oxygenase domains (red ovals) of two NOS monomers are usually in a dimeric form stabilized by the oxygenase (heme) domains. Top, in the absence of CaM, there is limited electron transfer through the NOS reductase domain (rectangles); NADPH reduces FAD via hydride transfer and then electrons are transferred from FAD to FMN. The electron transfer from FMN to the heme is impaired. Bottom, when CaM binds to the NOS it helps stabilize a conformation where the electron transfer from FMN to heme is enabled and the oxygenase domain can catalyze NO synthesis from L-Arginine. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] perspective (10). Reviews on H 4 B biochemistry have also treated NOS (11,12). In this review, we will focus on the function of tetrahydrobiopterin in NOS enzymes. H 4 B Requirement for NOS Catalysis Early studies of NOS enzymes identified H 4 B as a necessary cofactor for NO synthesis (13 15) with a stoichiometry of one molecule of H 4 B per subunit (16). Nevertheless, the function of H 4 B was controversial, with a variety of suggested roles. A redox H 4 B Binding in NOS Pterin Binding Affinity of NOS NOS enzymes bind H 4 B with high affinity. The presence of significant amounts of H 4 B in the enzyme after purification evinces the tight binding of the cofactor by the native protein (13 15). The binding of L-arginine and H 4 B shows a synergistic effect: prior binding of L-arginine increases the binding affinity for H 4 B and vice versa (21,29,30). Mammalian NOS enzymes bind H 4 B with affinities in the nm range. H 4 B is usually the preferred substrate over H 2 B, except for the enos enzyme (31). This fact has important implications for human pathology (10,12,31). Bacterial NOS enzymes operate in a wide range of K D values, and sometimes bind H 4 F with higher affinity (2,32,33). It should be noted that although many other pterins can bind to NOS (see (11) and references therein), only a few H 4 B analogs can support NO synthesis (21). The binding affinities for several pterins are shown in Table 1. Pterin Binding Pocket in Mammalian and Bacterial NOS Structures of the oxygenase domains for the three eukaryotic NOS isoforms (34 36) and several bacterial NOS proteins (37 39) have been reported. The alignment of the sequences for SCH 1 NOS converts L-Arginine to L-Citrulline and NO in a two-step process. Tejero and Stuehr 359

3 IUBMB LIFE TABLE 1 Dissociation constants for pterin binding to NOS enzymes Enzyme H 4 B(lM) H 4 F(lM) H 2 B(lM) þl-arg L-Arg þl-arg L-Arg References inos ND 6.2 (21) nnos 0.037, >10, (29,32) enos , ND (30,31) bsnos 0.1 ND 0.4 ND (32) deinos 10 ND 20 ND (33) Reported values are K D from radiolabeled pterin binding studies (29 31) or K M values from NO synthesis kinetics (21,32,33). inos, mouse inos; nnos, rat nnos; enos, bovine enos; bsnos, bacillus subtilis NOS; deinos, deinococcus radiodurans NOS; ND, not determined. FIG 2 Sequence alignment of mammalian and bacterial NOS proteins. Regions involved in dimerization are shown in yellow; Znbinding cysteines are shown in green; residues directly binding H 4 BorH 4 F are shown in red and blue to indicate that they belong to different monomers. Note the additional N-terminus structure for the mammalian NOS proteins, including the N-terminal hook region and the Zn binding motif. nnos, rat nnos; inos, mouse inos enos, bovine enos; bsnos, Bacillus subtilis NOS, sanos, Staphylococcus aureus NOS; gsnos, Geobacillus stearothermophilus NOS. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] 360 Tetrahydrobiopterin in NOS

4 FIG 3 Protein environment of the H 4 B/H 4 F cofactor. Panels A and B, comparison of the overall structures of inos (panel A) and bsnos (Panel B). Monomers are shown in blue and yellow. The extra N-terminal portion of inos, not present in bacterial NOS, is shown in red. Heme (pink), H 4 B(H 4 F for bsnos, yellow) and L-Arg (NOHA for bsnos, blue) are shown as sticks. Panels C and D, detail of the interactions of the H 4 B/H 4 F cofactor. Panel C, Several inos residues and the heme group form a hydrogen bonding network with the H 4 B cofactor. Relevant inos residues are shown in green and grey; H 4 B (yellow), Heme (pink), and the substrate L-Arginine (blue) are shown as sticks. Trp455 and Phe470 are shown in grey color to indicate that they belong to the other monomer. Two water molecules that form H-bonding interactions with H 4 B are shown as red spheres. The hydrogen bonding interactions are shown as yellow dashes. Ser112, Ile456, and Trp457 make H-bonds through the main chain carbonyl groups; Arg375 interacts through its side chain. The side chain of Trp457 forms a p-stacking interaction with H 4 B. Panel D, Several bsnos residues and the heme group form a hydrogen bonding network with the H 4 F cofactor. Relevant bsnos residues are shown in green and grey; H 4 F (yellow), Heme (pink), and the substrate NOHA (blue) are shown as sticks. Trp323 and Phe338 are shown in grey color to indicate that they belong to the other monomer. The hydrogen bonding interactions are shown as yellow dashes. Thr324 and Trp325 make H-bonds through the main chain carbonyl groups; Arg243 interacts through its side chain. The side chain of Trp325 forms a p-stacking interaction with H 4 F. The figure was made using PyMOL ( and the crystal structure of the mouse inosoxy dimer (PDB entry 1NOD (34)) and the bsnos dimer (PDB entry 1M7Z (38)). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] these NOS proteins indicates the notable similarity between mammalian and bacterial NOS enzymes (Fig. 2). The presence of additional elements in the N-termini of the mammalian proteins can be also noted; these motifs are involved in dimer formation and pterin binding (Figs. 2 and 3). The available structures clearly show that the absence of this N-termini allows bacterial NOS proteins to bind larger pterins (Fig. 3). The conservation of the H 4 B binding environments in enos, inos, and nnos is remarkable. Moreover, the bacterial NOS proteins also share many similarities in the pterin binding region. H 4 B is bound to NOS by an extensive network of hydrogen bonds, with a highly conserved pattern for the three NOS isoforms (34 36) (Fig. 3). Most hydrogen bonds are contributed by the protein but there is also an interaction with the heme group. Several hydrogen bonds are contributed by the protein backbone and the side chain of a conserved Arg residue (Arg375 in mouse inos). A conserved tryptophan residue also provides stabilization through a p-stacking interaction (Trp457 in mouse inos). Notably both Arg and Trp residues are conserved in mammalian and bacterial NOS proteins. The pterin binding pocket is placed in the oxygenase dimer interface. This location clearly suggest a relationship with the role of H 4 B binding on stabilization of the NOS dimer. H 4 B Function in NOS Catalysis H 4 B Role in the L-Arginine Hydroxylation Step Despite initial difficulties to assess the role of H 4 B in NOS catalysis, the study of the formation of NOHA from L-arginine Tejero and Stuehr 361

5 IUBMB LIFE FIG 4 Time course of the formation of the oxygen complex (Fe II O 2 ), oxidized heme (Fe III ), NOHA, and H 4 B radical during the single turnover of inos. Traces for Fe II O 2 and Fe III are determined by stopped-flow experiments; NOHA is determined from rapid-quench experiments; H 4 B radical is determined by Electron Paramagnetic Resonance on rapid freeze experiments. Data from (26). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] yielded increasing evidence of a role for H 4 B beyond the H 4 B H 2 B cycle of aromatic hydroxylases. The presence of H 4 B increases the rate of the Fe II O 2 complex decay, a novel role for H 4 B (20). Other studies observed that NOHA formation only occurred in the presence of H 4 B (40,41). As previously pointed out, the observation of a H 4 B radical formation during the reaction of NOS with L-arginine clearly indicated the active role of H 4 B during the formation of NOHA from L-arginine (23 26). The observation of the build-up of an H 4 B radical as the ferrous oxygen complex decays was instrumental in establishing a coherent mechanism (26) (Fig. 4). First steps of the reaction were clear, with the reductase domain of NOS providing the electrons to reduce the heme, and subsequent oxygen binding to form a ferrous-dioxygen complex (Fe II O 2 ). As this species decays with concomitant formation of an H 4 B radical, a one electron transfer from H 4 Bto the heme-oxy complex with the formation of a ferric peroxo species is expected. From this point, different reactions are possible. By analogy with P450 systems, protonation of this species to form the ferric-hydroperoxo species and eventual formation of the Fe IV ¼O porphyrin radical (Compound I) species was proposed (Fig. 5). In this process, the H 4 B radical is still present after L-arginine hydroxylation, and an additional electron provided by the flavoprotein domain (42) is needed to return to the initial resting state. H 4 B Role in the NOHA Oxidation Step The formation of citrulline and NO from NOHA has many similarities with the process of L-arginine oxidation to NOHA and also substantial differences. We must consider that the FIG 5 Putative mechanism for the hydroxylation of L-Arginine by NOS. Boxes in continuous lines indicate stable species, boxes in dotted lines indicate transient intermediate species. The oxidation state of H 4 B and the substrate bound (L-Arginine/NOHA) are shown in the top left and right, respectively, of each species. The transfer of an electron from H 4 B to the ferrous-oxy species triggers the formation of a ferric-peroxy species that will capture two protons and eliminate a water molecule to form a compound I-like species that will carry out the hydroxylation of the substrate L-Arginine. Note that the H 4 B radical (red) is not reduced after NOHA formation and has to receive one electron from the flavin domain to continue the catalytic cycle. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] 362 Tetrahydrobiopterin in NOS

6 FIG 6 Putative mechanism for the oxidation of NOHA to NO and L-citrulline by NOS. Boxes in continuous lines indicate stable species, boxes in dotted lines indicate transient intermediate species. The oxidation state of H 4 B and the substrate bound (NOHA/L-citrulline) are shown in the top left and right, respectively, of each species. The transfer of an electron from H 4 B to the ferrous-oxy species triggers the formation of a ferric peroxy species that will evolve to a hydroperoxo species that will react with NOHA to form citrulline and a ferrous nitroxyl species. Subsequent electron transfer from the ferrous nitroxyl species to the H 4 B radical will yield the observed Fe III NO species. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] oxidation of L-arginine to NOHA requires two electrons and two protons, but the reaction of NOHA to citrulline and NO requires one electron, as a two electron donation would lead to the formation of nitroxyl (NO ) species as final product. Therefore, it was considered that the electron transfer from H 4 B may not be necessary, and even detrimental, for this step. Extensive evidence further indicates that H 4 B is necessary for NOHA oxidation (43 45). Interestingly, reaction of nnos with NOHA and hydrogen peroxide in the absence of H 4 B leads to the formation of nitroxyl, whereas H 4 B directs the reaction toward NO instead (46). Therefore, it was proposed that the H 4 B radical formed in the first steps of the reaction could actually be reduced by a Fe II NO intermediate to produce the observed Fe III -NO species (24,46,47). Later reports are consistent with such model (44,48 50). These observations indicate that the requirement for H 4 B in the NOHA oxidation step appears to be two-pronged (Fig. 6). First, H 4 B it is needed to transfer an electron to the ferrous-dioxygen complex, as for the L-arginine hydroxylation step. Then, after the reaction of the ferric hydroperoxo species with NOHA, a ferrous nitroxyl complex is formed. The H 4 B radical will be reduced by this species giving the product ferric NO complex and regenerating the H 4 B. Effect of Mutations in the H 4 B Binding Pocket The effect of changes in the H 4 B binding pocket illustrates the fine tuning of the H 4 B reactivity in NOS. The mutation of inos Trp457 by Phe or Ala decreases the rate of H 4 B radical formation and accelerates its decay. The ferrous-dioxygen complex also decays at a slower rate similar to that of H 4 B radical formation, as expected from the mechanism. The amount of H 4 B radical stabilized by Trp457Phe and Trp457Ala is around 75% of wild-type enzyme. These changes cause a decreased NOHA yield (51). Similar effects are observed for the nnos mutants Trp678Phe and Trp678Ala, indicating a conserved role for this residue in the different isoforms (52). NOHA single turnover experiments have been also reported for the inos mutants Trp457Phe and Trp457Ala. The mutants show a decrease in the reaction rate, no detectable formation of H 4 B radical or Fe III NO species, and decreased citrulline yield (53). The mutants are able to produce NO, to a lesser extent than the wild-type enzyme. These observations suggest that the lack of Fe III NO species is due to a rate of formation slower than that of NO dissociation from the Fe III NO complex, unlike the wild type enzyme where enough Fe III NO accumulates to be detected. The mutations cause the Fe II O 2 complex to decay at a slower rate; this decrease correlates with a decreased citrulline yield. These results confirm that modification of the H 4 B radical stability impacts the reaction of H 4 Bwiththe Fe II O 2 complex in both reaction steps. Replacement of inos Arg375 by Lys, Asn, or Asp also causes a decrease in the reaction yield. The underlying causes appear more complex in this case. For the Arg375Asn and Arg375Asp, these effects are partly related to impaired formation of the NOS dimer and diminished ability to stabilize the H 4 B radical. Alternatively, the Arg375Lys mutant is able to form the H 4 B radical at a faster rate than wild-type inos. Tejero and Stuehr 363

7 IUBMB LIFE However, Arg375Lys showed lower NOHA yield, decreased NO synthesis, and increased NADPH oxidation. Thus, fast H 4 B reactions can lead to increased uncoupling of NADPH oxidation and NO synthesis. These results stress the need for a timely electron transfer for NO synthesis (54). Conclusions Our knowledge about H 4 B function in NOS has rapidly evolved. In less than 25 years, H 4 B has gone from an exotic cofactor of unknown function to a specialized electron donor with a role never seen before in H 4 B dependent enzymes. Some questions about H 4 B function still remain, the possible role of H 4 B as proton donor during catalysis or the exact protonation role of the H 4 B species is still discussed (8,9,28). The details of the reaction in bacterial NOS, although largely similar to mammalian NOS, can yield some surprises (2,45). References [1] Pfeiffer, S., Mayer, B., and Hemmens, B. (1999) Nitric oxide: chemical puzzles posed by a biological messenger. Angew. Chem. Int. 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