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High-pressure studies of the reaction mechanism of nitric-oxide synthase
Biochimica et Biophysica Acta 1764 (2006) 578 – 585 http://www.elsevier.com/locate/bba
Review
High-pressure studies of the reaction mechanism of nitric-oxide synthase Antonius C.F. Gorren b,*, Ste´phane Marchal a, Morten Sørlie c,d, K. Kristoffer Andersson c, Reinhard Lange a, Bernd Mayer b a INSERM U710, De´partement Biologie-Sante´, Universite´ Montpellier II, IFR 122, Montpellier, France Institut fu¨r Pharmakologie und Toxikologie, Karl-Franzens-Universita¨t Graz, Universita¨tsplatz 2, A-8010 Graz, Austria c Department of Molecular Biosciences, University of Oslo, PO Box 1041 Blindern, N-0316 Oslo, Norway Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, PO Box 5003, N-1432 A˚s, Norway b
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Received 3 October 2005; accepted 2 November 2005 Available online 28 November 2005
Abstract Nitric-oxide synthase (NOS) generates nitric oxide from l-arginine in two reaction cycles with N N-hydroxy-l-arginine as an obligate intermediate. Although much progress has been made in recent years in the elucidation of the reaction mechanism of NOS, many questions remain to be answered. The use of low temperature has been instrumental in the revelation of the mechanism of NO synthesis, particularly regarding the role of the cofactor 5,6,7,8-tetrahydrobopterin (BH4). High-pressure studies may be expected to be similarly useful, but have been very few so far. In this short review, we depict the present state of knowledge about the reaction mechanism of NO synthesis, and the role(s) BH4 plays in it. This exposition is followed by a summary of the results obtained thus far in high-pressure studies and of the conclusions that can be drawn from them. D 2005 Elsevier B.V. All rights reserved. Keywords: Nitric-oxide synthase; High pressure; Tetrahydrobiopterin; Heme ligand binding
1. Introduction Nitric oxide synthase (NOS, see Refs. [1– 10] for reviews) catalyzes the conversion of l-arginine (Arg) to l-citrulline and NO in two discrete reactions, with intermediate formation of Nhydroxy-l-arginine (NHA). Both reactions utilize molecular oxygen as a co-substrate and NADPH as an electron source. Whereas each reaction consumes one equivalent of O2, the electron stoichiometries are different, with net consumptions of 2 and 1 equivalent in the first and second reaction, respectively. NOS is a homodimer, with each subunit consisting of a CAbbreviations: NOS, nitric oxide synthase; eNOS, nNOS, and iNOS, endothelial, neuronal, and inducible isoforms of NOS; eNOSoxy, isolated oxygenase domain of (bovine) eNOS; NHA, N N-hydroxy-l-arginine; BH4, tetrahydrobiopterin ((6R)-5,6,7,8-tetrahydro-6-(l-erythro-1V,2V-dihydroxypropyl)pterin); BH3 , 6,7,8-trihydrobiopterin radical; BH2, 7,8-dihydrobiopterin; 4-Am-BH4, 4-amino-tetrahydrobiopterin ((6R)-2,4-diamino-5,6,7,8-tetrahydro6-(l-erythro-1V,2V-dihydroxypropyl)pteridine); 4-Am-BH3 and 4-Am-BH2, see BH3 and BH2; EPR, electron paramagnetic resonance; UV/Vis, ultraviolet visible * Corresponding author. Tel.: +43 316 380 5569; fax: +43 316 380 9890. E-mail address: [email protected] (A.C.F. Gorren).
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1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.11.005
terminal reductase domain and a N-terminal oxygenase domain with a calmodulin binding sequence in between. Catalysis takes place at a heme moiety in the oxygenase domain. Like the heme in cytochrome P450, NOS heme in its active form is 5coordinate high-spin with a cysteine thiolate for an axial ligand. Oxygen binds to the heme iron as the sixth ligand in the distal pocket, which also contains the binding site for substrate (Arg/ NHA). The electrons originate from NADPH via two flavin (FAD and FMN) moieties in the reductase domain, which is structurally related to cytochrome P450 reductase. Interdomain electron transfer requires the presence of calmodulin at its binding site. Extant hypotheses on the mechanism of NO synthesis are based on mechanism(s) proposed for similar cytochrome P450catalyzed reactions (for reviews on the mechanism of P450, see Refs. [11– 15]). The reaction cycle starts with reduction of the heme (sometimes preceded by substrate binding), followed by O2 binding. After a second reduction and two protonation steps, H2O is split off the complex, yielding the hydroxylating species, which, in the reaction with Arg, is believed to have a Compound I type structure. This species abstracts hydrogen from the substrate and, in the subsequent oxygen-rebound step, ferric
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heme is regenerated and the hydroxylated product (NHA) is formed. Because of the different electronic requirements it is generally assumed that the second reaction follows a different pathway, involving the direct interaction of the substrate NHA with the ferric (hydro)peroxo complex that is formed after the second reduction, without participation of a compound I type structure. It should be noted that little hard evidence exists for any steps occurring after the second reduction, and that the mechanistic details for subsequent steps are largely conjectured.
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seconds in single-turnover (and therefore easily observed by EPR spectroscopy [22,23]), since the electron is incorporated in the product (NHA). In the second cycle, with NHA as the substrate, formation of the pterin radical is very short-lived (tens of milliseconds [33]) since the electron is not incorporated in the product, allowing it to be transferred back to the pteridine after completion of the cycle. 4. BH4 as a key component of the proton relay pathway in NOS
2. The cofactor tetrahydrobiopterin (BH4) One striking difference between cytochrome P450 and NOS is that the latter entertains tetrahydrobiopterin (BH4) as an additional cofactor in close proximity to the heme (for recent comprehensive reviews on the role of BH4 in NO synthesis, see Refs. [7,16,17]). NOS cannot hydroxylate Arg and synthesize NO in the absence of BH4; instead, the NADPH-derived electrons are utilized to reduce O2 to O2 . BH4 was earlier identified as a cofactor in the aromatic amino acid hydroxylases, where it is directly involved in binding and reduction of oxygen, OUO bond splitting, and substrate hydroxylation, in concert with a non-heme iron [18 – 20]. In these enzymes, BH4 shuttles between the tetrahydro- and quinonoid dihydro- redox states, while acting as a dissociable cofactor. A similar function of BH4 in NOS was anticipated, but could soon be ruled out. BH4 has several structural and allosteric effects in NOS—it enhances substrate affinity, induces a low-to-high spin transition of the heme, and stimulates dimerization—that may have regulatory significance but cannot explain the absolute dependence of NO synthesis on the pterin cofactor. Despite the evidence against 2-electron redox shuttling, a redox function of BH4 was suggested by results obtained with BH4 analogues. In general, these studies demonstrated that tetrahydropteridines like BH4 were able to sustain NO synthesis, whereas 7,8dihydropteridines such as BH2 (7,8-dihydrobiopterin) could mimic all structural and allosteric effects of BH4, but did not support NO synthesis. Instead, these compounds acted as BH4competitive inhibitors and uncoupling agents, shifting to production of O2 instead of NO. 3. BH4 as a transient reductant of the oxyferrous complex On the basis of singe-turnover studies with a variety of techniques, including low-temperature and rapid-scan/stoppedflow UV/vis spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and product analysis, it was established that the central role of BH4 consists in the reduction of the oxyferrous complex [21 – 35]. It appears that, unlike for cytochrome P450, the Fe(II)O2 complex in NOS is so unstable that it decays to ferric heme and O2 before a second electron can be transferred from NADPH to the heme by the reductase domain [7,17,36]. The involvement of BH4 as an accessory 1electron donor prevents uncoupling by reducing the oxyferrous complex to Fe(II)O2 , thereby allowing the catalytic cycle to continue. In the first cycle with Arg as the substrate, the BH3 radical formed in this way is stable on a timescale of
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Whereas the function of BH4 as a transient 1-electron donor to the heme during NO synthesis is now widely accepted, a second essential catalytic role, recently attributed to BH4, is more controversial. This function was proposed on the basis of studies with the BH4 analog 4-amino-tetrahydrobiopterin (4Am-BH4), which is one of the best-documented and widely used pterin-based inhibitors of NOS [32,37 –43]. Although specifically designed as a potentially redox-inactive NOS inhibitor [37], paradoxical results were obtained in electrochemical studies. Against expectations, these studies showed that the electrochemical properties of 4-Am-BH4 were similar to those of BH4, while confirming the redox-inactivity of BH2 and other 7,8-dihydropteridines under physiologically relevant conditions [44]. This suggested that BH4 performed another function in NO synthesis than reduction of Fe(II)O2, and that this second essential function was inhibited in the presence of 4-Am-BH4. It had already been suggested, on the basis of X-ray crystallographic studies [45], that BH4 might be involved in one or both of the protonation steps that immediately follow the second reduction. The crystal structure of NOS suggests a proton transfer pathway from BH4, which is in direct contact with several water molecules in the substrate entrance cleft and hence with the bulk solvent, to the heme-bound ligand, involving only a propionate side chain on the heme porphyrin, the bound substrate molecule, and a strictly conserved glutamate residue [46]. Of special importance for this pathway is a hydrogen bond between N-3 of BH4 and the propionate of the porphyrin. The pK a values of N-3 for BH4 and 4-Am-BH4 are expected to be very different, with the most stable tautomers of BH4 and 4-Am-BH4 being protonated and deprotonated at this position, respectively. Therefore, it seemed plausible that the inhibitory properties of 4-Am-BH4 might be due to the inability of this pteridine to support protonation of the reduced oxyferrous complex. To investigate this possibility, low-temperature UV/vis absorption and EPR spectroscopic studies were carried out [46,47]. In the cycle with Arg, the 4-Am-BH3 radical was formed together with an enzyme species that was identified as the oxyferrous complex. In the cycle with NHA no radical accumulated, but a ferrous heme-NO complex was formed. These observations allowed following conclusions: (i) 4-AmBH4 is able to supplant BH4 as a 1-electron donor to the oxyferrous complex, in line with the electrochemical studies; (ii) with both substrates the heme was still reduced after the reaction, suggesting that the cycle is arrested at some stage between formation of Fe(II)O2 and regeneration of Fe(III). These results
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are consistent with the proposed role of BH4 (but not 4-AmBH4) as an essential component of the proton relay pathway. 5. Role of BH4 in the catalytic mechanism of NO synthesis The mechanism of NO synthesis by NOS is illustrated in Fig. 1, which highlights the well-established role of BH4 as a 1-electron donor, as well as its more speculative function in protonation. Accordingly, rapid completion of the reaction cycle is only possible in the presence of BH4; in its absence, or in the presence of redox-inactive pteridines like BH2, decay of the oxyferrous complex results in almost complete uncoupling of catalysis. With 4-Am-BH4, reduction of the oxyferrous complex can take place, but uncoupling nevertheless ensues, because the lack of protonation of the reduced oxyferrous complex results in formation of O2 with regeneration of Fe(III) and 4-Am-BH4. 6. High-pressure studies of NOS: comparison of O2 and CO binding High pressure can and has been used as a tool in the elucidation of enzyme reaction mechanisms (for a recent review on the use of high pressure in enzymatic studies, see Ref. [48]). We have commenced to apply high-pressure
Fig. 2. Comparison of the pressure dependence of the reaction of CO and O2 with reduced eNOSoxy. Plotted are the observed pseudo-first order rates of CO binding (closed circles), and of formation (open squares) and disappearance (closed diamonds) of the O2 complex in the presence of Arg (0.5 mM) and 4Am-BH2 (50 AM).
Fig. 1. Role of BH4 in NO synthesis. Illustration of the hypothetical reaction cycle of NOS. The reaction cycles with Arg and NHA are identical at least until formation of Fe(II)O2H, after which they may diverge. BH4 is proposed to be involved as a 1-electron donor to Fe(II)O2 and as an integral part of the proton conductance chain required for protonation of Fe(II)O2 (and, not shown in the figure, of Fe(II)O2H as well, at least in the first reaction cycle). Please note that although in the figure BH4 is designated as a proton donor for convenience, it is neither the immediate donor (which is the substrate) nor the ultimate donor (which is the solvent). In the absence of pterin or with redox-inactive pteridines uncoupling results in ferric heme and superoxide. In the presence of 4-AmBH4, the ferrous-superoxy complex can and will be formed, but uncoupling yields the same result as without pterin, because, after O2 dissociation, ferrous heme will reduce the pterin radical, resulting in regeneration of Fe(III) and 4Am-BH4. In the presence of BH4 but without substrate, uncoupling results in direct formation of H2O2.
stopped-flow UV/vis spectroscopy to study the binding of oxygen to the reduced heme of the isolated oxygenase domain of endothelial NOS under various conditions. A similar approach has been used successfully with cytochrome P450 [49]. However, for NOS, the oxyferrous complex is so unstable that no useful information can be obtained by this method under physiological conditions, i.e., in the presence of substrate and BH4. Often, CO and O2 binding are very similar, with the important difference that the CO complex is not processed any further. Therefore, we investigated if CO binding could be used as a model for O2 binding in the case of NOS. To do so, we compared binding of both ligands under conditions where the oxy complex is relatively stable. It has been found that the stability of the oxyferrous complex is greatly increased (by two orders of magnitude) in the presence of inhibitory pteridines [24,43,50]. Instead of the archetypal BH2, we used 4-Am-BH2 (4-amino-7,8-dihydrobiopterin) for better comparison with its reduced counterpart 4-Am-BH4, in which we have a special interest (see above). As can be see from Fig. 2, CO binding was very different from the O2 reaction in the presence of Arg and 4-Am-BH2, being considerably slower (7– 8) at atmospheric pressure, and exhibiting a different dependence on pressure [51]. Apparently, CO binding is a poor model for the reaction with O2.
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7. Effects of substrate on CO and O2 binding To search for the origin of the remarkable differences between CO and O2 binding, both reactions were studied in the presence of the substrates Arg and NHA and the inhibitory pteridines 4-Am-BH4 and 4-Am-BH2 in all possible combinations [51]. CO binding was not affected by the identity of the substrate and the pteridine at all, suggesting that the observed reaction step is not the actual binding of CO to the heme, but takes place outside of the heme pocket. Binding of O2, by contrast, was clearly affected when Arg was replaced by NHA (Fig. 3). Although the observed binding rates were similar at atmospheric pressure, the pressure dependence was totally different, with increasing and decreasing rates at higher pressures for NHA and Arg, respectively. The decay of the O2 complex was affected by the substrate as well, as the lifetime of the oxycomplex with Arg was almost pressureindependent, whereas the stability of the complex in the presence of NHA was markedly improved at high pressure (Fig. 3). These results suggest that the reaction step observed here does take place in the active site pocket and most likely represents the binding of O2 to the heme.
Fig. 4. Effect of Arg on the pressure dependence of CO binding to reduced rat nNOS. Plotted are the observed pseudo-first order rates of CO binding in the absence (closed circles), and presence (open squares) of Arg (0.2 mM). The enzyme preparation contained approximately 1 equivalent of BH4 per NOS dimer; no exogenous BH4 was added.
Clues about the origin of the slow binding of CO can be found in earlier studies with full-length neuronal NOS [52], which gave similar results to those discussed above in the presence of Arg, but much faster binding that was virtually insensitive to pressure in its absence (Fig. 4). A striking decrease in CO binding rate in the presence of Arg was observed in stopped-flow and flash-photolysis studies with different NOS isoforms as well [53 – 55]. These results suggest strong inhibition of CO binding by Arg that is relieved at high pressure by expulsion of the substrate [52]. In line with that assumption, rates of CO binding in the absence of Arg in those studies were comparable to the rates of O2 binding in the studies discussed above. A conceivable explanation for the observations is illustrated in Fig. 5A. Accordingly, the rate of CO binding is determined by a slow initial binding step to an unidentified site outside of the heme pocket that is followed by rapid transfer of the ligand to the heme. With O2, this early binding step is either not necessary or much faster than with CO. However, alternative explanations are also possible, for instance if CO binding, but not O2 binding requires a conformational change of the protein (Fig. 5B), or if Arg has to move from its preferred binding site before CO (but not O2) can bind (Fig. 5C). The latter hypothesis would agree well with the results illustrated in Fig. 4. Interpretation is complicated by the possible generation of both 5- and 6-coordinate complexes [53]. Although more data will be required to resolve this issue, the inadequacy of CO binding as a model for the reaction with oxygen is obvious. 8. Effects of pteridines on O2 binding Fig. 3. Effect of substrate on the pressure dependence of the reaction of O2 with reduced eNOSoxy. Plotted are the observed pseudo-first order rates of the formation (circles) and disappearance (squares) of the O2 complex in the presence of 4-Am-BH2 (50 AM) and 0.5 mM Arg (open symbols) or 1 mM NHA (closed symbols).
Whereas exchanging the pteridine cofactor did not affect CO binding (see above), it markedly affected formation and stability of the oxyferrous complex (Fig. 6). As already noted (see above), O2 binding slowed down with increasing pressure in the
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O2 binding was even more pronounced [51]. Binding in the presence of 4-Am-BH4 was much slower than with 4-Am-BH2 at atmospheric pressure, but the rate increase as a function of pressure was much steeper. As with Arg, there was a clear break in the pressure dependence of O2 binding between 40 and 60 MPa, with moderately decreasing rates at higher pressures. The pronounced differences between 4-Am-BH4 and 4-AmBH2 are more surprising than the effects of the substrate discussed above, since the pteridine cofactor is not bound in the active site pocket, and although binding of the pteridine per se will have strong allosteric effects on the heme pocket and, hence, on ligand binding [7], such effects are expected to be very similar for all pteridines [45]. It is therefore likely that the observed differences correspond to an immediate effect of the pteridine(s) on the reaction. In this respect, it may be significant that with 4-Am-BH4, but not with 4-Am-BH2, the pressure dependence of O2 binding exhibited break points that might be indicative of changes in the rate limiting step. Since 4-Am-BH4, but not 4-Am-BH2 is able to reduce the oxyferrous complex [46,56], one is tempted to interpret the break in the pressure dependence by a shift from rate-limiting formation of
Fig. 5. Putative explanations for the differences between CO and O2 binding. According to hypothesis A, CO binds to a site outside of the actual heme pocket in a slow early step, which is followed by a faster transfer to the heme. With O2, the early binding step is not necessary or very fast. Alternatively, CO binding may require a thermodynamically disfavored NOS conformation (hypothesis B) or Arg binding mode (hypothesis C). In all cases, it is the entry and exit of CO rather than CO binding to the heme that is impeded by Arg or an Arg/BH4-induced conformational change, since the effects are of a kinetic rather than thermodynamic nature.
presence of 4-Am-BH2 and Arg; by contrast, with 4-Am-BH4 the rate of O2 binding became faster up to a pressure of approximately 50 MPa, and decreased above that pressure [51]. Decay of the complex was similar for 4-Am-BH4 and 4-AmBH2, except for a consistently higher rate in the presence of 4Am-BH4. In the presence of NHA, the effect of the pteridine on
Fig. 6. Effect of pteridines on the pressure dependence of the reaction of O2 with reduced eNOSoxy. Plotted are the observed pseudo-first order rates of the formation (circles) and disappearance (squares) of the O2 complex in the presence of 0.5 mM Arg (formation: circles, decay: diamonds) or 1 mM NHA (formation: squares, decay: triangles) and 50 AM of either 4-Am-BH2 (open symbols, dashed lines) and 4-Am-BH4 (closed symbols, continuous lines).
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Fe(II)O2 to rate-limiting formation of Fe(II)O2 (due to an increase of the rate of Fe(II)O2 formation beyond the rate of Fe(II)O2 reduction above ¨50 MPa). However, that would imply that the intermediate observed at atmospheric pressure be the Fe(II)O2 species, which conflicts with previous spectroscopic results [46,50]. Alternatively, decay of the ferroussuperoxy species could become slower than its formation at 50 MPa, but in that case the break should be in the pressure dependence of the decay rather than the formation of the oxycomplex. Moreover, in the case of a change in rate-limiting step, the pressure at which the break occurs would be determined by the thermodynamic parameters of the various reactions, which differ between the two substrates. This implies that only chance could cause the coincidence of the pressure break points with both substrates if a change in rate-limiting step is involved, which rather suggests that changes in the protein structure may be at the root of the observed phenomena instead. Further studies will be necessary to bring light into these issues. 9. High-pressure and internal electron transfer Another research topic for which high-pressure studies were conducted concerns the regulation of NOS internal electron transfer by calmodulin [52]. Calmodulin is required for interdomain electron transfer from FMN to the heme. Cytochrome c is known as an artificial electron acceptor for NOS that reacts only with the FMN cofactor [57,58]. Although this reaction occurs in the absence of calmodulin, it is stimulated some 20-fold in its presence. In studies with the neuronal isoform, high pressure was shown to stimulate the NOS-catalyzed reduction of cytochrome c by NADPH in the absence of calmodulin to an extent that surpassed the calmodulin-stimulated activity above 100 MPa [52]. Since similar effects could also be triggered in the presence of chaotropic agents [52,59], these observations suggested that partial unfolding is the driving force. These results, therefore, fit in well with the now generally accepted hypothesis that an autoinhibitory loop, present in the FMN binding domain of the constitutive isoforms neuronal and endothelial NOS (nNOS and eNOS), but not in inducible NOS (iNOS) and cytochrome P450 reductase, blocks interdomain electron transfer in the constitutive isoforms and that binding of calmodulin to these isoforms relieves inhibition by displacing the loop from its binding site [60,61]. 10. Outlook In addition to allowing the determination of thermodynamic parameters like the reaction and activation volumes, highpressure studies sometimes offer the opportunity to observe and identify intermediates that are invisible by other means for kinetic reasons [48]. In this respect, high-pressure studies resemble studies at various temperatures, but are more versatile, since high-pressure studies are not restricted by the fluidity of the solvent and do not require the presence of a cryosolvent [28,47,49]. Low-temperature studies have been
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instrumental in the elucidation of the molecular mechanism of NO synthesis [21,23,24,62], raising hopes that high-pressure studies may turn out equally prolific. As is clear from the data discussed above, we are still at the very beginning of highpressure investigations of NOS catalysis, and even within the limited scope of the studies undertaken until now much work still needs to be done. In particular, the spectral characteristics of the observed intermediates in the reaction with O2 need to be determined to assess whether the observed pressure breaks with 4-Am-BH4 [51] correspond to a change in rate-limiting step. Other pteridines must be tried out, to corroborate the assumption that the large effect on exchanging 4-Am-BH2 with 4-Am-BH4 derives from a property particular to 4-AmBH4. Studies of NO binding may be useful, both on their own account since NO complexes are believed to be intermediates in the catalytic cycle [3,6 – 9,23,24,63,64], and also to evaluate if NO binding provides a better-suited model for the reaction with O2 than CO binding turned out to be. Nevertheless, even from the few data gathered so far [51,52], it appears that highpressure studies of NOS catalysis may prove a valuable tool in the further characterization of the mechanism of NO synthesis. Acknowledgments Experimental studies in the authors’ laboratories were supported by the Fonds zur Fo¨rderung der Wissenschaftlichen ¨ sterreich and by a grant (RGP0026/2001-M) Forschung in O from the Human Frontier Science Project. References [1] S. Pfeiffer, B. Mayer, B. Hemmens, Nitric oxide: chemical puzzles posed by a biological messenger, Angew. Chem. Int. Ed. 38 (1999) 1714 – 1731. [2] P.J. Andrew, B. Mayer, Enzymatic function of nitric oxide synthases, Cardiovasc. Res. 43 (1999) 521 – 531. [3] D.J. Stuehr, Mammalian nitric oxide synthases, Biochim. Biophys. Acta 1411 (1999) 217 – 230. [4] M.L. Ludwig, M.A. Marletta, A new decoration for nitric oxide synthase—A Zn(Cys)4 site, Structure 7 (1999) R73 – R79. [5] J.T. Groves, C.C.-Y. Wang, Nitric oxide synthase: models and mechanisms, Curr. Opin. Chem. Biol. 4 (2000) 687 – 695. [6] W.K. Alderton, C.E. Cooper, R.G. Knowles, Nitric oxide synthases: structure, function, and inhibition, Biochem. J. 357 (2001) 593 – 615. [7] A.C.F. Gorren, B. Mayer, Tetrahydrobiopterin in nitric oxide synthesis: a novel biological role for pteridines, Curr. Drug Metab. 3 (2002) 133 – 157. [8] D.J. Stuehr, J. Santolini, Z.-Q. Wang, C.-C. Wei, S. Adak, Update on mechanism and catalytic regulation in the NO synthases, J. Biol. Chem. 279 (2004) 36167 – 36170. [9] D.L. Rousseau, D. Li, M. Couture, S.-R. Yeh, Ligand – protein interactions in nitric oxide synthase, J. Inorg. Biochem. 99 (2005) 306 – 323. [10] H. Li, T.L. Poulos, Structure – function studies on nitric oxide synthases, J. Inorg. Biochem. 99 (2005) 293 – 305. [11] R.E. White, M.J. Coon, Oxygen activation by cytochrome P-450, Annu. Rev. Biochem. 49 (1980) 315 – 356. [12] E.J. Mueller, P.J. Loida, S.G. Sligar, Twenty-five years of P450cam research, in: P.R. Ortiz de Montellano (Ed.), Cytochrome P450: Structure, Mechanism, and Biochemistry, 2nd edR, Plenum Press, New York, 1995, pp. 83 – 124. [13] M. Sono, M.P. Roach, E.D. Coulter, J.H. Dawson, Heme-containing oxygenases, Chem. Rev. 96 (1996) 2841 – 2887.
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[14] B. Meunier, S.P. de Visser, S. Shaik, Mechanism of oxidation reactions catalyzed by cytochrome P450 enzymes, Chem. Rev. 104 (2004) 3947 – 3980. [15] I.G. Denisov, T.M. Makris, S.G. Sligar, I. Schlichting, Structure and chemistry of cytochrome P450, Chem. Rev. 105 (2005) 2253 – 2277. [16] E.R. Werner, A.C.F. Gorren, R. Heller, G. Werner-Felmayer, B. Mayer, Tetrahydrobiopterin and nitric oxide: mechanistic and pharmacological aspects, Exp. Biol. Med. 228 (2003) 1291 – 1302. [17] C.-C. Wei, B.R. Crane, D.J. Stuehr, Tetrahydrobiopterin radical enzymology, Chem. Rev. 103 (2003) 2365 – 2383. [18] T.J. Kappock, J.P. Caradonna, Pterin-dependent amino acid hydroxylases, Chem. Rev. 96 (1996) 2659 – 2756. [19] P.F. Fitzpatrick, Tetrahydropterin-dependent amino acid hydroxylases, Annu. Rev. Biochem. 68 (1999) 355 – 381. [20] P.F. Fitzpatrick, Mechanism of aromatic amino acid hydroxylation, Biochemistry 42 (2003) 14083 – 14091. [21] N. Bec, A.C.F. Gorren, C. Voelker, B. Mayer, R. Lange, Reaction of neuronal nitric-oxide synthase with oxygen at low temperature, J. Biol. Chem. 273 (1998) 13502 – 13508. [22] A.R. Hurshman, C. Krebs, D.E. Edmondson, B.H. Huynh, M.A. Marletta, Formation of a pterin radical in the reaction of the heme domain of inducible nitric oxide synthase with oxygen, Biochemistry 38 (1999) 15689 – 15696. [23] N. Bec, A.C.F. Gorren, B. Mayer, P.P. Schmidt, K.K. Andersson, R. Lange, The role of tetrahydrobiopterin in the activation of oxygen by nitric-oxide synthase, J. Inorg. Biochem. 81 (2000) 207 – 211. [24] A.C.F. Gorren, N. Bec, A. Schrammel, E.R. Werner, R. Lange, B. Mayer, Low-temperature optical absorption spectra suggest a redox role for tetrahydrobiopterin in both steps of nitric oxide synthase catalysis, Biochemistry 39 (2000) 11763 – 11770. [25] P.P. Schmidt, R. Lange, A.C.F. Gorren, E.R. Werner, B. Mayer, K.K. Andersson, Formation of a protonated trihydrobiopterin radical cation in the first reaction cycle of neuronal and endothelial nitric oxide synthase detected by electron paramagnetic resonance spectroscopy, J. Biol. Inorg. Chem. 6 (2001) 151 – 158. [26] C.-C. Wei, Z.-Q. Wang, Q. Wang, A.L. Meade, C. Hemann, R. Hille, D.J. Stuehr, Rapid kinetic studies link tetrahydrobiopterin radical formation to heme-dioxy reduction and arginine hydroxylation in inducible nitric-oxide synthase, J. Biol. Chem. 276 (2001) 315 – 319. [27] C.-C. Wei, Z.-Q. Wang, D.J. Stuehr, Nitric oxide synthase: use of stoppedflow spectroscopy and rapid-quench methods in single-turnover conditions to examine formation and reactions of heme-O2 intermediate in early catalysis, Methods Enzymol. 354 (2002) 320 – 338. [28] A.C.F. Gorren, N. Bec, R. Lange, B. Mayer, Redox role for tetrahydrobiopterin in nitric oxide synthase catalysis: low-temperature optical absorption spectral detection, Methods Enzymol. 353 (2002) 114 – 121. [29] A.R. Hurshman, M.A. Marletta, Reactions catalyzed by the heme domain of inducible nitric oxide synthase: evidence for the involvement of tetrahydrobiopterin in electron transfer, Biochemistry 41 (2002) 3439 – 3456. [30] M. Du, H.-C. Yeh, V. Berka, L.-H. Wang, A. Tsai, Redox properties of human endothelial nitric-oxide synthase oxygenase and reductase domains purified from yeast expression system, J. Biol. Chem. 278 (2003) 6002 – 6011. [31] C.-C. Wei, Z.-Q. Wang, A.S. Arvai, C. Hemann, R. Hille, E.D. Getzoff, D.J. Stuehr, Structure of tetrahydrobiopterin tunes its electron transfer to the heme-dioxy intermediate in nitric oxide synthase, Biochemistry 42 (2003) 1969 – 1977. [32] A.R. Hurshman, C. Krebs, D.E. Edmondson, M.A. Marletta, Ability of tetrahydrobiopterin analogues to support catalysis by inducible nitric oxide synthase: formation of a pterin radical is required for enzyme activity, Biochemistry 42 (2003) 13287 – 13303. [33] C.-C. Wei, Z.-Q. Wang, C. Hemann, R. Hille, D.J. Stuehr, A tetrahydrobiopterin radical forms and then becomes reduced during N Nhydroxyarginine oxidation by nitric-oxide synthase, J. Biol. Chem. 278 (2003) 46668 – 46673.
[34] V. Berka, H.-C. Yeh, D. Gao, F. Kiran, A.-L. Tsai, Redox function of tetrahydrobiopterin and effect of l-arginine on oxygen binding in endothelial nitric oxide synthase, Biochemistry 43 (2004) 13137 – 13148. [35] C.-C. Wei, Z.-Q. Wang, D. Durra, C. Hemann, R. Hille, E.D. Garcin, E.D. Getzoff, D.J. Stuehr, The three nitric-oxide synthases differ in their kinetics of tetrahydrobiopterin radical formation, heme-dioxy reduction, and arginine hydroxylation, J. Biol. Chem. 280 (2005) 8929 – 8935. [36] C.-C. Wei, Z.-Q. Wang, A.L. Meade, J.F. McDonald, D.J. Stuehr, Why do nitric oxide synthases use tetrahydrobiopterin? J. Inorg. Biochem. 91 (2002) 618 – 624. [37] E.R. Werner, E. Pitters, K. Schmidt, H. Wachter, G. Werner-Felmayer, B. Mayer, Identification of the 4-amino analogue of tetrahydrobiopterin as a dihydropteridine reductase inhibitor and a potent pteridine antagonist of rat neuronal nitric oxide synthase, Biochem. J. 320 (1996) 193 – 196. [38] S. Pfeiffer, A.C.F. Gorren, E. Pitters, K. Schmidt, E.R. Werner, B. Mayer, Allosteric regulation of rat brain nitric oxide synthase by the pterin-site enzyme inhibitor 4-aminotetrahydrobiopterin, Biochem. J. 328 (1997) 349 – 352. [39] B. Mayer, C. Wu, A.C.F. Gorren, S. Pfeiffer, K. Schmidt, P. Clark, D.J. Stuehr, E.R. Werner, Tetrahydrobiopterin binding to macrophage inducible nitric oxide synthase: heme spin shift and dimer stabilization by the potent pterin antagonist 4-amino-tetrahydrobiopterin, Biochemistry 36 (1997) 8422 – 8427. [40] H.M. Bo¨mmel, A. Reif, L.G. Fro¨hlich, A. Frey, H. Hofmann, D.M. Marecak, V. Groehn, P. Kotsonis, M. La, S. Ko¨ster, M. Meinecke, M. Bernhardt, M. Weeger, S. Ghisla, G.D. Prestwich, W. Pfleiderer, H.H.H.W. Schmidt, Anti-pterins as tools to characterize the function of tetrahydrobiopterin in NO synthase, J. Biol. Chem. 273 (1998) 33142 – 33149. [41] A. Reif, L.G. Fro¨hlich, P. Kotsonis, A. Frey, H.M. Bo¨mmel, D.A. Wink, W. Pfleiderer, H.H.H.W. Schmidt, Tetrahydrobiopterin inhibits monomerization and is consumed during catalysis in neuronal NO synthase, J. Biol. Chem. 274 (1999) 24921 – 24929. [42] E.R. Werner, G. Werner-Felmayer, Biopterin analogues: novel nitric oxide synthase inhibitors with immunosuppressive action, Curr. Drug Metab. 3 (2002) 119 – 121. [43] T.W.B. Ost, S. Daff, Thermodynamic and kinetic analysis of the nitrosyl, carbonyl, and dioxy heme complexes of neuronal nitric-oxide synthase, J. Biol. Chem. 280 (2005) 965 – 973. [44] A.C.F. Gorren, A.J. Kungl, K. Schmidt, E.R. Werner, B. Mayer, Electrochemistry of pterin cofactors and inhibitors of nitric oxide synthase, Nitric Oxide 5 (2001) 176 – 186. [45] B.R. Crane, A.S. Arvai, S. Ghosh, E.D. Getzoff, D.J. Stuehr, J.A. Tainer, Structures of the N N-hydroxy-l-arginine complex of inducible nitric oxide synthase oxygenase dimer with active and inactive pterins, Biochemistry 39 (2000) 4608 – 4621. [46] M. Sørlie, A.C.F. Gorren, S. Marchal, T. Shimizu, R. Lange, K.K. Andersson, B. Mayer, Single-turnover of nitric-oxide synthase in the presence of 4-amino-tetrahydrobiopterin, J. Biol. Chem. 278 (2003) 48602 – 48610. [47] A.C.F. Gorren, M. Sørlie, K.K. Andersson, S. Marchal, R. Lange, B. Mayer, Tetrahydrobiopterin as combined electron/proton donor in nitric oxide biosynthesis: cryogenic UV-vis and EPR detection of reaction intermediates, Methods Enzymol. 396 (2005) 456 – 466. [48] P. Masson, C. Balny, Linear and non-linear pressure dependence of enzyme catalytic parameters, Biochim. Biophys. Acta 1724 (2005) 440 – 450. [49] F. Bancel, G. Hui Bon Hoa, P. Anzenbacher, C. Balny, R. Lange, High pressure: a new tool to study P450 structure and function, Methods Enzymol. 357 (2002) 145 – 157. [50] S. Marchal, A.C.F. Gorren, M. Sørlie, K.K. Andersson, B. Mayer, R. Lange, Evidence of two distinct oxygen complexes of reduced endothelial nitric oxide synthase, J. Biol. Chem. 279 (2004) 19824 – 19831. [51] S. Marchal, H.M. Girvan, A.C.F. Gorren, B. Mayer, A.W. Munro, C. Balny, R. Lange, Formation of transient oxygen complexes of cytochrome P450 BM3 and nitric oxide synthase under high pressure, Biophys. J. 85 (2003) 3303 – 3309.
A.C.F. Gorren et al. / Biochimica et Biophysica Acta 1764 (2006) 578 – 585 [52] R. Lange, N. Bec, P. Anzenbacher, A.W. Munro, A.C.F. Gorren, B. Mayer, Use of high pressure to study elementary steps in P450 and nitric oxide synthase, J. Inorg. Biochem. 87 (2001) 191 – 195. [53] H.M. Abu-Soud, C. Wu, D.K. Ghosh, D.J. Stuehr, Stopped-flow analysis of CO and NO binding to inducible nitric oxide synthase, Biochemistry 37 (1998) 3777 – 3786. [54] J.S. Scheele, E. Bruner, T. Zemojtel, P. Marta´sek, L.J. Roman, B.S.S. Masters, V.S. Sharma, D. Magde, Kinetics of CO and NO ligation with the Cys331YAla mutant of neuronal nitric-oxide synthase, J. Biol. Chem. 276 (2001) 4733 – 4736. [55] T.H. Stevenson, A.F. Gutierrez, W.K. Alderton, L. Lian, N.S. Scrutton, Kinetics of CO binding to the haem domain of murine inducible nitric oxide synthase: differential effects of haem domain ligands, Biochem. J. 358 (2001) 201 – 208. [56] S. Marchal, R. Lange, M. Sørlie, K.K. Andersson, A.C.F. Gorren, B. Mayer, CO exchange of the oxyferrous complexes of endothelial nitricoxide synthase oxygenase domain in the presence of 4-amino-tetrahydrobiopterin, J. Inorg. Biochem. 98 (2004) 1217 – 1222. [57] R.G. Knowles, S. Moncada, Nitric oxide synthases in mammals, Biochem. J. 298 (1994) 249 – 258. [58] O.W. Griffith, D.J. Stuehr, Nitric oxide synthases: properties and catalytic mechanism, Annu. Rev. Physiol. 57 (1995) 707 – 736.
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[59] R. Narayanasami, J.S. Nishimura, K. McMillan, L.J. Roman, T.M. Shea, A.M. Robida, P.M. Horowitz, B.S.S. Masters, The influence of chaotropic reagents on neuronal nitric oxide synthase and its flavoprotein module. Urea and guanidine hydrochloride stimulate NADPH-cytochrome c reductase activity of both proteins, Nitric Oxide 1 (1997) 39 – 49. [60] P. Lane, S.S. Gross, The autoinhibitory control element and calmodulin conspire to provide physiological modulation of endothelial and neuronal nitric oxide synthase activity, Acta Physiol. Scand. 168 (2000) 53 – 63. [61] L.J. Roman, P. Marta´sek, B.S.S. Masters, Intrinsic and extrinsic modulation of nitric oxide synthase activity, Chem. Rev. 102 (2002) 1179 – 1189. [62] A.P. Ledbetter, K. McMillan, L.J. Roman, B.S.S. Masters, J.H. Dawson, M. Sono, Low-temperature stabilization and spectroscopic characterization of the dioxygen complex of the ferrous neuronal nitric oxide synthase oxygenase domain, Biochemistry 38 (1999) 8014 – 8021. [63] H.M. Abu-Soud, J. Wang, D.L. Rousseau, J.M. Fukuto, L.J. Ignarro, D.J. Stuehr, Neuronal nitric oxide synthase self-inactivates by forming a ferrous-nitrosyl complex during aerobic catalysis, J. Biol. Chem. 270 (1995) 22997 – 23006. [64] J. Santolini, S. Adak, C.M.L. Curran, D.J. Stuehr, A kinetic simulation model that describes catalysis and regulation in nitric-oxide synthase, J. Biol. Chem. 276 (2001) 1233 – 1243.
Review
High-pressure studies of the reaction mechanism of nitric-oxide synthase Antonius C.F. Gorren b,*, Ste´phane Marchal a, Morten Sørlie c,d, K. Kristoffer Andersson c, Reinhard Lange a, Bernd Mayer b a INSERM U710, De´partement Biologie-Sante´, Universite´ Montpellier II, IFR 122, Montpellier, France Institut fu¨r Pharmakologie und Toxikologie, Karl-Franzens-Universita¨t Graz, Universita¨tsplatz 2, A-8010 Graz, Austria c Department of Molecular Biosciences, University of Oslo, PO Box 1041 Blindern, N-0316 Oslo, Norway Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, PO Box 5003, N-1432 A˚s, Norway b
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Received 3 October 2005; accepted 2 November 2005 Available online 28 November 2005
Abstract Nitric-oxide synthase (NOS) generates nitric oxide from l-arginine in two reaction cycles with N N-hydroxy-l-arginine as an obligate intermediate. Although much progress has been made in recent years in the elucidation of the reaction mechanism of NOS, many questions remain to be answered. The use of low temperature has been instrumental in the revelation of the mechanism of NO synthesis, particularly regarding the role of the cofactor 5,6,7,8-tetrahydrobopterin (BH4). High-pressure studies may be expected to be similarly useful, but have been very few so far. In this short review, we depict the present state of knowledge about the reaction mechanism of NO synthesis, and the role(s) BH4 plays in it. This exposition is followed by a summary of the results obtained thus far in high-pressure studies and of the conclusions that can be drawn from them. D 2005 Elsevier B.V. All rights reserved. Keywords: Nitric-oxide synthase; High pressure; Tetrahydrobiopterin; Heme ligand binding
1. Introduction Nitric oxide synthase (NOS, see Refs. [1– 10] for reviews) catalyzes the conversion of l-arginine (Arg) to l-citrulline and NO in two discrete reactions, with intermediate formation of Nhydroxy-l-arginine (NHA). Both reactions utilize molecular oxygen as a co-substrate and NADPH as an electron source. Whereas each reaction consumes one equivalent of O2, the electron stoichiometries are different, with net consumptions of 2 and 1 equivalent in the first and second reaction, respectively. NOS is a homodimer, with each subunit consisting of a CAbbreviations: NOS, nitric oxide synthase; eNOS, nNOS, and iNOS, endothelial, neuronal, and inducible isoforms of NOS; eNOSoxy, isolated oxygenase domain of (bovine) eNOS; NHA, N N-hydroxy-l-arginine; BH4, tetrahydrobiopterin ((6R)-5,6,7,8-tetrahydro-6-(l-erythro-1V,2V-dihydroxypropyl)pterin); BH3 , 6,7,8-trihydrobiopterin radical; BH2, 7,8-dihydrobiopterin; 4-Am-BH4, 4-amino-tetrahydrobiopterin ((6R)-2,4-diamino-5,6,7,8-tetrahydro6-(l-erythro-1V,2V-dihydroxypropyl)pteridine); 4-Am-BH3 and 4-Am-BH2, see BH3 and BH2; EPR, electron paramagnetic resonance; UV/Vis, ultraviolet visible * Corresponding author. Tel.: +43 316 380 5569; fax: +43 316 380 9890. E-mail address: [email protected] (A.C.F. Gorren).
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1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.11.005
terminal reductase domain and a N-terminal oxygenase domain with a calmodulin binding sequence in between. Catalysis takes place at a heme moiety in the oxygenase domain. Like the heme in cytochrome P450, NOS heme in its active form is 5coordinate high-spin with a cysteine thiolate for an axial ligand. Oxygen binds to the heme iron as the sixth ligand in the distal pocket, which also contains the binding site for substrate (Arg/ NHA). The electrons originate from NADPH via two flavin (FAD and FMN) moieties in the reductase domain, which is structurally related to cytochrome P450 reductase. Interdomain electron transfer requires the presence of calmodulin at its binding site. Extant hypotheses on the mechanism of NO synthesis are based on mechanism(s) proposed for similar cytochrome P450catalyzed reactions (for reviews on the mechanism of P450, see Refs. [11– 15]). The reaction cycle starts with reduction of the heme (sometimes preceded by substrate binding), followed by O2 binding. After a second reduction and two protonation steps, H2O is split off the complex, yielding the hydroxylating species, which, in the reaction with Arg, is believed to have a Compound I type structure. This species abstracts hydrogen from the substrate and, in the subsequent oxygen-rebound step, ferric
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heme is regenerated and the hydroxylated product (NHA) is formed. Because of the different electronic requirements it is generally assumed that the second reaction follows a different pathway, involving the direct interaction of the substrate NHA with the ferric (hydro)peroxo complex that is formed after the second reduction, without participation of a compound I type structure. It should be noted that little hard evidence exists for any steps occurring after the second reduction, and that the mechanistic details for subsequent steps are largely conjectured.
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seconds in single-turnover (and therefore easily observed by EPR spectroscopy [22,23]), since the electron is incorporated in the product (NHA). In the second cycle, with NHA as the substrate, formation of the pterin radical is very short-lived (tens of milliseconds [33]) since the electron is not incorporated in the product, allowing it to be transferred back to the pteridine after completion of the cycle. 4. BH4 as a key component of the proton relay pathway in NOS
2. The cofactor tetrahydrobiopterin (BH4) One striking difference between cytochrome P450 and NOS is that the latter entertains tetrahydrobiopterin (BH4) as an additional cofactor in close proximity to the heme (for recent comprehensive reviews on the role of BH4 in NO synthesis, see Refs. [7,16,17]). NOS cannot hydroxylate Arg and synthesize NO in the absence of BH4; instead, the NADPH-derived electrons are utilized to reduce O2 to O2 . BH4 was earlier identified as a cofactor in the aromatic amino acid hydroxylases, where it is directly involved in binding and reduction of oxygen, OUO bond splitting, and substrate hydroxylation, in concert with a non-heme iron [18 – 20]. In these enzymes, BH4 shuttles between the tetrahydro- and quinonoid dihydro- redox states, while acting as a dissociable cofactor. A similar function of BH4 in NOS was anticipated, but could soon be ruled out. BH4 has several structural and allosteric effects in NOS—it enhances substrate affinity, induces a low-to-high spin transition of the heme, and stimulates dimerization—that may have regulatory significance but cannot explain the absolute dependence of NO synthesis on the pterin cofactor. Despite the evidence against 2-electron redox shuttling, a redox function of BH4 was suggested by results obtained with BH4 analogues. In general, these studies demonstrated that tetrahydropteridines like BH4 were able to sustain NO synthesis, whereas 7,8dihydropteridines such as BH2 (7,8-dihydrobiopterin) could mimic all structural and allosteric effects of BH4, but did not support NO synthesis. Instead, these compounds acted as BH4competitive inhibitors and uncoupling agents, shifting to production of O2 instead of NO. 3. BH4 as a transient reductant of the oxyferrous complex On the basis of singe-turnover studies with a variety of techniques, including low-temperature and rapid-scan/stoppedflow UV/vis spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and product analysis, it was established that the central role of BH4 consists in the reduction of the oxyferrous complex [21 – 35]. It appears that, unlike for cytochrome P450, the Fe(II)O2 complex in NOS is so unstable that it decays to ferric heme and O2 before a second electron can be transferred from NADPH to the heme by the reductase domain [7,17,36]. The involvement of BH4 as an accessory 1electron donor prevents uncoupling by reducing the oxyferrous complex to Fe(II)O2 , thereby allowing the catalytic cycle to continue. In the first cycle with Arg as the substrate, the BH3 radical formed in this way is stable on a timescale of
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Whereas the function of BH4 as a transient 1-electron donor to the heme during NO synthesis is now widely accepted, a second essential catalytic role, recently attributed to BH4, is more controversial. This function was proposed on the basis of studies with the BH4 analog 4-amino-tetrahydrobiopterin (4Am-BH4), which is one of the best-documented and widely used pterin-based inhibitors of NOS [32,37 –43]. Although specifically designed as a potentially redox-inactive NOS inhibitor [37], paradoxical results were obtained in electrochemical studies. Against expectations, these studies showed that the electrochemical properties of 4-Am-BH4 were similar to those of BH4, while confirming the redox-inactivity of BH2 and other 7,8-dihydropteridines under physiologically relevant conditions [44]. This suggested that BH4 performed another function in NO synthesis than reduction of Fe(II)O2, and that this second essential function was inhibited in the presence of 4-Am-BH4. It had already been suggested, on the basis of X-ray crystallographic studies [45], that BH4 might be involved in one or both of the protonation steps that immediately follow the second reduction. The crystal structure of NOS suggests a proton transfer pathway from BH4, which is in direct contact with several water molecules in the substrate entrance cleft and hence with the bulk solvent, to the heme-bound ligand, involving only a propionate side chain on the heme porphyrin, the bound substrate molecule, and a strictly conserved glutamate residue [46]. Of special importance for this pathway is a hydrogen bond between N-3 of BH4 and the propionate of the porphyrin. The pK a values of N-3 for BH4 and 4-Am-BH4 are expected to be very different, with the most stable tautomers of BH4 and 4-Am-BH4 being protonated and deprotonated at this position, respectively. Therefore, it seemed plausible that the inhibitory properties of 4-Am-BH4 might be due to the inability of this pteridine to support protonation of the reduced oxyferrous complex. To investigate this possibility, low-temperature UV/vis absorption and EPR spectroscopic studies were carried out [46,47]. In the cycle with Arg, the 4-Am-BH3 radical was formed together with an enzyme species that was identified as the oxyferrous complex. In the cycle with NHA no radical accumulated, but a ferrous heme-NO complex was formed. These observations allowed following conclusions: (i) 4-AmBH4 is able to supplant BH4 as a 1-electron donor to the oxyferrous complex, in line with the electrochemical studies; (ii) with both substrates the heme was still reduced after the reaction, suggesting that the cycle is arrested at some stage between formation of Fe(II)O2 and regeneration of Fe(III). These results
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are consistent with the proposed role of BH4 (but not 4-AmBH4) as an essential component of the proton relay pathway. 5. Role of BH4 in the catalytic mechanism of NO synthesis The mechanism of NO synthesis by NOS is illustrated in Fig. 1, which highlights the well-established role of BH4 as a 1-electron donor, as well as its more speculative function in protonation. Accordingly, rapid completion of the reaction cycle is only possible in the presence of BH4; in its absence, or in the presence of redox-inactive pteridines like BH2, decay of the oxyferrous complex results in almost complete uncoupling of catalysis. With 4-Am-BH4, reduction of the oxyferrous complex can take place, but uncoupling nevertheless ensues, because the lack of protonation of the reduced oxyferrous complex results in formation of O2 with regeneration of Fe(III) and 4-Am-BH4. 6. High-pressure studies of NOS: comparison of O2 and CO binding High pressure can and has been used as a tool in the elucidation of enzyme reaction mechanisms (for a recent review on the use of high pressure in enzymatic studies, see Ref. [48]). We have commenced to apply high-pressure
Fig. 2. Comparison of the pressure dependence of the reaction of CO and O2 with reduced eNOSoxy. Plotted are the observed pseudo-first order rates of CO binding (closed circles), and of formation (open squares) and disappearance (closed diamonds) of the O2 complex in the presence of Arg (0.5 mM) and 4Am-BH2 (50 AM).
Fig. 1. Role of BH4 in NO synthesis. Illustration of the hypothetical reaction cycle of NOS. The reaction cycles with Arg and NHA are identical at least until formation of Fe(II)O2H, after which they may diverge. BH4 is proposed to be involved as a 1-electron donor to Fe(II)O2 and as an integral part of the proton conductance chain required for protonation of Fe(II)O2 (and, not shown in the figure, of Fe(II)O2H as well, at least in the first reaction cycle). Please note that although in the figure BH4 is designated as a proton donor for convenience, it is neither the immediate donor (which is the substrate) nor the ultimate donor (which is the solvent). In the absence of pterin or with redox-inactive pteridines uncoupling results in ferric heme and superoxide. In the presence of 4-AmBH4, the ferrous-superoxy complex can and will be formed, but uncoupling yields the same result as without pterin, because, after O2 dissociation, ferrous heme will reduce the pterin radical, resulting in regeneration of Fe(III) and 4Am-BH4. In the presence of BH4 but without substrate, uncoupling results in direct formation of H2O2.
stopped-flow UV/vis spectroscopy to study the binding of oxygen to the reduced heme of the isolated oxygenase domain of endothelial NOS under various conditions. A similar approach has been used successfully with cytochrome P450 [49]. However, for NOS, the oxyferrous complex is so unstable that no useful information can be obtained by this method under physiological conditions, i.e., in the presence of substrate and BH4. Often, CO and O2 binding are very similar, with the important difference that the CO complex is not processed any further. Therefore, we investigated if CO binding could be used as a model for O2 binding in the case of NOS. To do so, we compared binding of both ligands under conditions where the oxy complex is relatively stable. It has been found that the stability of the oxyferrous complex is greatly increased (by two orders of magnitude) in the presence of inhibitory pteridines [24,43,50]. Instead of the archetypal BH2, we used 4-Am-BH2 (4-amino-7,8-dihydrobiopterin) for better comparison with its reduced counterpart 4-Am-BH4, in which we have a special interest (see above). As can be see from Fig. 2, CO binding was very different from the O2 reaction in the presence of Arg and 4-Am-BH2, being considerably slower (7– 8) at atmospheric pressure, and exhibiting a different dependence on pressure [51]. Apparently, CO binding is a poor model for the reaction with O2.
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7. Effects of substrate on CO and O2 binding To search for the origin of the remarkable differences between CO and O2 binding, both reactions were studied in the presence of the substrates Arg and NHA and the inhibitory pteridines 4-Am-BH4 and 4-Am-BH2 in all possible combinations [51]. CO binding was not affected by the identity of the substrate and the pteridine at all, suggesting that the observed reaction step is not the actual binding of CO to the heme, but takes place outside of the heme pocket. Binding of O2, by contrast, was clearly affected when Arg was replaced by NHA (Fig. 3). Although the observed binding rates were similar at atmospheric pressure, the pressure dependence was totally different, with increasing and decreasing rates at higher pressures for NHA and Arg, respectively. The decay of the O2 complex was affected by the substrate as well, as the lifetime of the oxycomplex with Arg was almost pressureindependent, whereas the stability of the complex in the presence of NHA was markedly improved at high pressure (Fig. 3). These results suggest that the reaction step observed here does take place in the active site pocket and most likely represents the binding of O2 to the heme.
Fig. 4. Effect of Arg on the pressure dependence of CO binding to reduced rat nNOS. Plotted are the observed pseudo-first order rates of CO binding in the absence (closed circles), and presence (open squares) of Arg (0.2 mM). The enzyme preparation contained approximately 1 equivalent of BH4 per NOS dimer; no exogenous BH4 was added.
Clues about the origin of the slow binding of CO can be found in earlier studies with full-length neuronal NOS [52], which gave similar results to those discussed above in the presence of Arg, but much faster binding that was virtually insensitive to pressure in its absence (Fig. 4). A striking decrease in CO binding rate in the presence of Arg was observed in stopped-flow and flash-photolysis studies with different NOS isoforms as well [53 – 55]. These results suggest strong inhibition of CO binding by Arg that is relieved at high pressure by expulsion of the substrate [52]. In line with that assumption, rates of CO binding in the absence of Arg in those studies were comparable to the rates of O2 binding in the studies discussed above. A conceivable explanation for the observations is illustrated in Fig. 5A. Accordingly, the rate of CO binding is determined by a slow initial binding step to an unidentified site outside of the heme pocket that is followed by rapid transfer of the ligand to the heme. With O2, this early binding step is either not necessary or much faster than with CO. However, alternative explanations are also possible, for instance if CO binding, but not O2 binding requires a conformational change of the protein (Fig. 5B), or if Arg has to move from its preferred binding site before CO (but not O2) can bind (Fig. 5C). The latter hypothesis would agree well with the results illustrated in Fig. 4. Interpretation is complicated by the possible generation of both 5- and 6-coordinate complexes [53]. Although more data will be required to resolve this issue, the inadequacy of CO binding as a model for the reaction with oxygen is obvious. 8. Effects of pteridines on O2 binding Fig. 3. Effect of substrate on the pressure dependence of the reaction of O2 with reduced eNOSoxy. Plotted are the observed pseudo-first order rates of the formation (circles) and disappearance (squares) of the O2 complex in the presence of 4-Am-BH2 (50 AM) and 0.5 mM Arg (open symbols) or 1 mM NHA (closed symbols).
Whereas exchanging the pteridine cofactor did not affect CO binding (see above), it markedly affected formation and stability of the oxyferrous complex (Fig. 6). As already noted (see above), O2 binding slowed down with increasing pressure in the
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O2 binding was even more pronounced [51]. Binding in the presence of 4-Am-BH4 was much slower than with 4-Am-BH2 at atmospheric pressure, but the rate increase as a function of pressure was much steeper. As with Arg, there was a clear break in the pressure dependence of O2 binding between 40 and 60 MPa, with moderately decreasing rates at higher pressures. The pronounced differences between 4-Am-BH4 and 4-AmBH2 are more surprising than the effects of the substrate discussed above, since the pteridine cofactor is not bound in the active site pocket, and although binding of the pteridine per se will have strong allosteric effects on the heme pocket and, hence, on ligand binding [7], such effects are expected to be very similar for all pteridines [45]. It is therefore likely that the observed differences correspond to an immediate effect of the pteridine(s) on the reaction. In this respect, it may be significant that with 4-Am-BH4, but not with 4-Am-BH2, the pressure dependence of O2 binding exhibited break points that might be indicative of changes in the rate limiting step. Since 4-Am-BH4, but not 4-Am-BH2 is able to reduce the oxyferrous complex [46,56], one is tempted to interpret the break in the pressure dependence by a shift from rate-limiting formation of
Fig. 5. Putative explanations for the differences between CO and O2 binding. According to hypothesis A, CO binds to a site outside of the actual heme pocket in a slow early step, which is followed by a faster transfer to the heme. With O2, the early binding step is not necessary or very fast. Alternatively, CO binding may require a thermodynamically disfavored NOS conformation (hypothesis B) or Arg binding mode (hypothesis C). In all cases, it is the entry and exit of CO rather than CO binding to the heme that is impeded by Arg or an Arg/BH4-induced conformational change, since the effects are of a kinetic rather than thermodynamic nature.
presence of 4-Am-BH2 and Arg; by contrast, with 4-Am-BH4 the rate of O2 binding became faster up to a pressure of approximately 50 MPa, and decreased above that pressure [51]. Decay of the complex was similar for 4-Am-BH4 and 4-AmBH2, except for a consistently higher rate in the presence of 4Am-BH4. In the presence of NHA, the effect of the pteridine on
Fig. 6. Effect of pteridines on the pressure dependence of the reaction of O2 with reduced eNOSoxy. Plotted are the observed pseudo-first order rates of the formation (circles) and disappearance (squares) of the O2 complex in the presence of 0.5 mM Arg (formation: circles, decay: diamonds) or 1 mM NHA (formation: squares, decay: triangles) and 50 AM of either 4-Am-BH2 (open symbols, dashed lines) and 4-Am-BH4 (closed symbols, continuous lines).
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Fe(II)O2 to rate-limiting formation of Fe(II)O2 (due to an increase of the rate of Fe(II)O2 formation beyond the rate of Fe(II)O2 reduction above ¨50 MPa). However, that would imply that the intermediate observed at atmospheric pressure be the Fe(II)O2 species, which conflicts with previous spectroscopic results [46,50]. Alternatively, decay of the ferroussuperoxy species could become slower than its formation at 50 MPa, but in that case the break should be in the pressure dependence of the decay rather than the formation of the oxycomplex. Moreover, in the case of a change in rate-limiting step, the pressure at which the break occurs would be determined by the thermodynamic parameters of the various reactions, which differ between the two substrates. This implies that only chance could cause the coincidence of the pressure break points with both substrates if a change in rate-limiting step is involved, which rather suggests that changes in the protein structure may be at the root of the observed phenomena instead. Further studies will be necessary to bring light into these issues. 9. High-pressure and internal electron transfer Another research topic for which high-pressure studies were conducted concerns the regulation of NOS internal electron transfer by calmodulin [52]. Calmodulin is required for interdomain electron transfer from FMN to the heme. Cytochrome c is known as an artificial electron acceptor for NOS that reacts only with the FMN cofactor [57,58]. Although this reaction occurs in the absence of calmodulin, it is stimulated some 20-fold in its presence. In studies with the neuronal isoform, high pressure was shown to stimulate the NOS-catalyzed reduction of cytochrome c by NADPH in the absence of calmodulin to an extent that surpassed the calmodulin-stimulated activity above 100 MPa [52]. Since similar effects could also be triggered in the presence of chaotropic agents [52,59], these observations suggested that partial unfolding is the driving force. These results, therefore, fit in well with the now generally accepted hypothesis that an autoinhibitory loop, present in the FMN binding domain of the constitutive isoforms neuronal and endothelial NOS (nNOS and eNOS), but not in inducible NOS (iNOS) and cytochrome P450 reductase, blocks interdomain electron transfer in the constitutive isoforms and that binding of calmodulin to these isoforms relieves inhibition by displacing the loop from its binding site [60,61]. 10. Outlook In addition to allowing the determination of thermodynamic parameters like the reaction and activation volumes, highpressure studies sometimes offer the opportunity to observe and identify intermediates that are invisible by other means for kinetic reasons [48]. In this respect, high-pressure studies resemble studies at various temperatures, but are more versatile, since high-pressure studies are not restricted by the fluidity of the solvent and do not require the presence of a cryosolvent [28,47,49]. Low-temperature studies have been
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instrumental in the elucidation of the molecular mechanism of NO synthesis [21,23,24,62], raising hopes that high-pressure studies may turn out equally prolific. As is clear from the data discussed above, we are still at the very beginning of highpressure investigations of NOS catalysis, and even within the limited scope of the studies undertaken until now much work still needs to be done. In particular, the spectral characteristics of the observed intermediates in the reaction with O2 need to be determined to assess whether the observed pressure breaks with 4-Am-BH4 [51] correspond to a change in rate-limiting step. Other pteridines must be tried out, to corroborate the assumption that the large effect on exchanging 4-Am-BH2 with 4-Am-BH4 derives from a property particular to 4-AmBH4. Studies of NO binding may be useful, both on their own account since NO complexes are believed to be intermediates in the catalytic cycle [3,6 – 9,23,24,63,64], and also to evaluate if NO binding provides a better-suited model for the reaction with O2 than CO binding turned out to be. Nevertheless, even from the few data gathered so far [51,52], it appears that highpressure studies of NOS catalysis may prove a valuable tool in the further characterization of the mechanism of NO synthesis. Acknowledgments Experimental studies in the authors’ laboratories were supported by the Fonds zur Fo¨rderung der Wissenschaftlichen ¨ sterreich and by a grant (RGP0026/2001-M) Forschung in O from the Human Frontier Science Project. References [1] S. Pfeiffer, B. Mayer, B. Hemmens, Nitric oxide: chemical puzzles posed by a biological messenger, Angew. Chem. Int. Ed. 38 (1999) 1714 – 1731. [2] P.J. Andrew, B. Mayer, Enzymatic function of nitric oxide synthases, Cardiovasc. Res. 43 (1999) 521 – 531. [3] D.J. Stuehr, Mammalian nitric oxide synthases, Biochim. Biophys. Acta 1411 (1999) 217 – 230. [4] M.L. Ludwig, M.A. Marletta, A new decoration for nitric oxide synthase—A Zn(Cys)4 site, Structure 7 (1999) R73 – R79. [5] J.T. Groves, C.C.-Y. Wang, Nitric oxide synthase: models and mechanisms, Curr. Opin. Chem. Biol. 4 (2000) 687 – 695. [6] W.K. Alderton, C.E. Cooper, R.G. Knowles, Nitric oxide synthases: structure, function, and inhibition, Biochem. J. 357 (2001) 593 – 615. [7] A.C.F. Gorren, B. Mayer, Tetrahydrobiopterin in nitric oxide synthesis: a novel biological role for pteridines, Curr. Drug Metab. 3 (2002) 133 – 157. [8] D.J. Stuehr, J. Santolini, Z.-Q. Wang, C.-C. Wei, S. Adak, Update on mechanism and catalytic regulation in the NO synthases, J. Biol. Chem. 279 (2004) 36167 – 36170. [9] D.L. Rousseau, D. Li, M. Couture, S.-R. Yeh, Ligand – protein interactions in nitric oxide synthase, J. Inorg. Biochem. 99 (2005) 306 – 323. [10] H. Li, T.L. Poulos, Structure – function studies on nitric oxide synthases, J. Inorg. Biochem. 99 (2005) 293 – 305. [11] R.E. White, M.J. Coon, Oxygen activation by cytochrome P-450, Annu. Rev. Biochem. 49 (1980) 315 – 356. [12] E.J. Mueller, P.J. Loida, S.G. Sligar, Twenty-five years of P450cam research, in: P.R. Ortiz de Montellano (Ed.), Cytochrome P450: Structure, Mechanism, and Biochemistry, 2nd edR, Plenum Press, New York, 1995, pp. 83 – 124. [13] M. Sono, M.P. Roach, E.D. Coulter, J.H. Dawson, Heme-containing oxygenases, Chem. Rev. 96 (1996) 2841 – 2887.
584
A.C.F. Gorren et al. / Biochimica et Biophysica Acta 1764 (2006) 578 – 585
[14] B. Meunier, S.P. de Visser, S. Shaik, Mechanism of oxidation reactions catalyzed by cytochrome P450 enzymes, Chem. Rev. 104 (2004) 3947 – 3980. [15] I.G. Denisov, T.M. Makris, S.G. Sligar, I. Schlichting, Structure and chemistry of cytochrome P450, Chem. Rev. 105 (2005) 2253 – 2277. [16] E.R. Werner, A.C.F. Gorren, R. Heller, G. Werner-Felmayer, B. Mayer, Tetrahydrobiopterin and nitric oxide: mechanistic and pharmacological aspects, Exp. Biol. Med. 228 (2003) 1291 – 1302. [17] C.-C. Wei, B.R. Crane, D.J. Stuehr, Tetrahydrobiopterin radical enzymology, Chem. Rev. 103 (2003) 2365 – 2383. [18] T.J. Kappock, J.P. Caradonna, Pterin-dependent amino acid hydroxylases, Chem. Rev. 96 (1996) 2659 – 2756. [19] P.F. Fitzpatrick, Tetrahydropterin-dependent amino acid hydroxylases, Annu. Rev. Biochem. 68 (1999) 355 – 381. [20] P.F. Fitzpatrick, Mechanism of aromatic amino acid hydroxylation, Biochemistry 42 (2003) 14083 – 14091. [21] N. Bec, A.C.F. Gorren, C. Voelker, B. Mayer, R. Lange, Reaction of neuronal nitric-oxide synthase with oxygen at low temperature, J. Biol. Chem. 273 (1998) 13502 – 13508. [22] A.R. Hurshman, C. Krebs, D.E. Edmondson, B.H. Huynh, M.A. Marletta, Formation of a pterin radical in the reaction of the heme domain of inducible nitric oxide synthase with oxygen, Biochemistry 38 (1999) 15689 – 15696. [23] N. Bec, A.C.F. Gorren, B. Mayer, P.P. Schmidt, K.K. Andersson, R. Lange, The role of tetrahydrobiopterin in the activation of oxygen by nitric-oxide synthase, J. Inorg. Biochem. 81 (2000) 207 – 211. [24] A.C.F. Gorren, N. Bec, A. Schrammel, E.R. Werner, R. Lange, B. Mayer, Low-temperature optical absorption spectra suggest a redox role for tetrahydrobiopterin in both steps of nitric oxide synthase catalysis, Biochemistry 39 (2000) 11763 – 11770. [25] P.P. Schmidt, R. Lange, A.C.F. Gorren, E.R. Werner, B. Mayer, K.K. Andersson, Formation of a protonated trihydrobiopterin radical cation in the first reaction cycle of neuronal and endothelial nitric oxide synthase detected by electron paramagnetic resonance spectroscopy, J. Biol. Inorg. Chem. 6 (2001) 151 – 158. [26] C.-C. Wei, Z.-Q. Wang, Q. Wang, A.L. Meade, C. Hemann, R. Hille, D.J. Stuehr, Rapid kinetic studies link tetrahydrobiopterin radical formation to heme-dioxy reduction and arginine hydroxylation in inducible nitric-oxide synthase, J. Biol. Chem. 276 (2001) 315 – 319. [27] C.-C. Wei, Z.-Q. Wang, D.J. Stuehr, Nitric oxide synthase: use of stoppedflow spectroscopy and rapid-quench methods in single-turnover conditions to examine formation and reactions of heme-O2 intermediate in early catalysis, Methods Enzymol. 354 (2002) 320 – 338. [28] A.C.F. Gorren, N. Bec, R. Lange, B. Mayer, Redox role for tetrahydrobiopterin in nitric oxide synthase catalysis: low-temperature optical absorption spectral detection, Methods Enzymol. 353 (2002) 114 – 121. [29] A.R. Hurshman, M.A. Marletta, Reactions catalyzed by the heme domain of inducible nitric oxide synthase: evidence for the involvement of tetrahydrobiopterin in electron transfer, Biochemistry 41 (2002) 3439 – 3456. [30] M. Du, H.-C. Yeh, V. Berka, L.-H. Wang, A. Tsai, Redox properties of human endothelial nitric-oxide synthase oxygenase and reductase domains purified from yeast expression system, J. Biol. Chem. 278 (2003) 6002 – 6011. [31] C.-C. Wei, Z.-Q. Wang, A.S. Arvai, C. Hemann, R. Hille, E.D. Getzoff, D.J. Stuehr, Structure of tetrahydrobiopterin tunes its electron transfer to the heme-dioxy intermediate in nitric oxide synthase, Biochemistry 42 (2003) 1969 – 1977. [32] A.R. Hurshman, C. Krebs, D.E. Edmondson, M.A. Marletta, Ability of tetrahydrobiopterin analogues to support catalysis by inducible nitric oxide synthase: formation of a pterin radical is required for enzyme activity, Biochemistry 42 (2003) 13287 – 13303. [33] C.-C. Wei, Z.-Q. Wang, C. Hemann, R. Hille, D.J. Stuehr, A tetrahydrobiopterin radical forms and then becomes reduced during N Nhydroxyarginine oxidation by nitric-oxide synthase, J. Biol. Chem. 278 (2003) 46668 – 46673.
[34] V. Berka, H.-C. Yeh, D. Gao, F. Kiran, A.-L. Tsai, Redox function of tetrahydrobiopterin and effect of l-arginine on oxygen binding in endothelial nitric oxide synthase, Biochemistry 43 (2004) 13137 – 13148. [35] C.-C. Wei, Z.-Q. Wang, D. Durra, C. Hemann, R. Hille, E.D. Garcin, E.D. Getzoff, D.J. Stuehr, The three nitric-oxide synthases differ in their kinetics of tetrahydrobiopterin radical formation, heme-dioxy reduction, and arginine hydroxylation, J. Biol. Chem. 280 (2005) 8929 – 8935. [36] C.-C. Wei, Z.-Q. Wang, A.L. Meade, J.F. McDonald, D.J. Stuehr, Why do nitric oxide synthases use tetrahydrobiopterin? J. Inorg. Biochem. 91 (2002) 618 – 624. [37] E.R. Werner, E. Pitters, K. Schmidt, H. Wachter, G. Werner-Felmayer, B. Mayer, Identification of the 4-amino analogue of tetrahydrobiopterin as a dihydropteridine reductase inhibitor and a potent pteridine antagonist of rat neuronal nitric oxide synthase, Biochem. J. 320 (1996) 193 – 196. [38] S. Pfeiffer, A.C.F. Gorren, E. Pitters, K. Schmidt, E.R. Werner, B. Mayer, Allosteric regulation of rat brain nitric oxide synthase by the pterin-site enzyme inhibitor 4-aminotetrahydrobiopterin, Biochem. J. 328 (1997) 349 – 352. [39] B. Mayer, C. Wu, A.C.F. Gorren, S. Pfeiffer, K. Schmidt, P. Clark, D.J. Stuehr, E.R. Werner, Tetrahydrobiopterin binding to macrophage inducible nitric oxide synthase: heme spin shift and dimer stabilization by the potent pterin antagonist 4-amino-tetrahydrobiopterin, Biochemistry 36 (1997) 8422 – 8427. [40] H.M. Bo¨mmel, A. Reif, L.G. Fro¨hlich, A. Frey, H. Hofmann, D.M. Marecak, V. Groehn, P. Kotsonis, M. La, S. Ko¨ster, M. Meinecke, M. Bernhardt, M. Weeger, S. Ghisla, G.D. Prestwich, W. Pfleiderer, H.H.H.W. Schmidt, Anti-pterins as tools to characterize the function of tetrahydrobiopterin in NO synthase, J. Biol. Chem. 273 (1998) 33142 – 33149. [41] A. Reif, L.G. Fro¨hlich, P. Kotsonis, A. Frey, H.M. Bo¨mmel, D.A. Wink, W. Pfleiderer, H.H.H.W. Schmidt, Tetrahydrobiopterin inhibits monomerization and is consumed during catalysis in neuronal NO synthase, J. Biol. Chem. 274 (1999) 24921 – 24929. [42] E.R. Werner, G. Werner-Felmayer, Biopterin analogues: novel nitric oxide synthase inhibitors with immunosuppressive action, Curr. Drug Metab. 3 (2002) 119 – 121. [43] T.W.B. Ost, S. Daff, Thermodynamic and kinetic analysis of the nitrosyl, carbonyl, and dioxy heme complexes of neuronal nitric-oxide synthase, J. Biol. Chem. 280 (2005) 965 – 973. [44] A.C.F. Gorren, A.J. Kungl, K. Schmidt, E.R. Werner, B. Mayer, Electrochemistry of pterin cofactors and inhibitors of nitric oxide synthase, Nitric Oxide 5 (2001) 176 – 186. [45] B.R. Crane, A.S. Arvai, S. Ghosh, E.D. Getzoff, D.J. Stuehr, J.A. Tainer, Structures of the N N-hydroxy-l-arginine complex of inducible nitric oxide synthase oxygenase dimer with active and inactive pterins, Biochemistry 39 (2000) 4608 – 4621. [46] M. Sørlie, A.C.F. Gorren, S. Marchal, T. Shimizu, R. Lange, K.K. Andersson, B. Mayer, Single-turnover of nitric-oxide synthase in the presence of 4-amino-tetrahydrobiopterin, J. Biol. Chem. 278 (2003) 48602 – 48610. [47] A.C.F. Gorren, M. Sørlie, K.K. Andersson, S. Marchal, R. Lange, B. Mayer, Tetrahydrobiopterin as combined electron/proton donor in nitric oxide biosynthesis: cryogenic UV-vis and EPR detection of reaction intermediates, Methods Enzymol. 396 (2005) 456 – 466. [48] P. Masson, C. Balny, Linear and non-linear pressure dependence of enzyme catalytic parameters, Biochim. Biophys. Acta 1724 (2005) 440 – 450. [49] F. Bancel, G. Hui Bon Hoa, P. Anzenbacher, C. Balny, R. Lange, High pressure: a new tool to study P450 structure and function, Methods Enzymol. 357 (2002) 145 – 157. [50] S. Marchal, A.C.F. Gorren, M. Sørlie, K.K. Andersson, B. Mayer, R. Lange, Evidence of two distinct oxygen complexes of reduced endothelial nitric oxide synthase, J. Biol. Chem. 279 (2004) 19824 – 19831. [51] S. Marchal, H.M. Girvan, A.C.F. Gorren, B. Mayer, A.W. Munro, C. Balny, R. Lange, Formation of transient oxygen complexes of cytochrome P450 BM3 and nitric oxide synthase under high pressure, Biophys. J. 85 (2003) 3303 – 3309.
A.C.F. Gorren et al. / Biochimica et Biophysica Acta 1764 (2006) 578 – 585 [52] R. Lange, N. Bec, P. Anzenbacher, A.W. Munro, A.C.F. Gorren, B. Mayer, Use of high pressure to study elementary steps in P450 and nitric oxide synthase, J. Inorg. Biochem. 87 (2001) 191 – 195. [53] H.M. Abu-Soud, C. Wu, D.K. Ghosh, D.J. Stuehr, Stopped-flow analysis of CO and NO binding to inducible nitric oxide synthase, Biochemistry 37 (1998) 3777 – 3786. [54] J.S. Scheele, E. Bruner, T. Zemojtel, P. Marta´sek, L.J. Roman, B.S.S. Masters, V.S. Sharma, D. Magde, Kinetics of CO and NO ligation with the Cys331YAla mutant of neuronal nitric-oxide synthase, J. Biol. Chem. 276 (2001) 4733 – 4736. [55] T.H. Stevenson, A.F. Gutierrez, W.K. Alderton, L. Lian, N.S. Scrutton, Kinetics of CO binding to the haem domain of murine inducible nitric oxide synthase: differential effects of haem domain ligands, Biochem. J. 358 (2001) 201 – 208. [56] S. Marchal, R. Lange, M. Sørlie, K.K. Andersson, A.C.F. Gorren, B. Mayer, CO exchange of the oxyferrous complexes of endothelial nitricoxide synthase oxygenase domain in the presence of 4-amino-tetrahydrobiopterin, J. Inorg. Biochem. 98 (2004) 1217 – 1222. [57] R.G. Knowles, S. Moncada, Nitric oxide synthases in mammals, Biochem. J. 298 (1994) 249 – 258. [58] O.W. Griffith, D.J. Stuehr, Nitric oxide synthases: properties and catalytic mechanism, Annu. Rev. Physiol. 57 (1995) 707 – 736.
585
[59] R. Narayanasami, J.S. Nishimura, K. McMillan, L.J. Roman, T.M. Shea, A.M. Robida, P.M. Horowitz, B.S.S. Masters, The influence of chaotropic reagents on neuronal nitric oxide synthase and its flavoprotein module. Urea and guanidine hydrochloride stimulate NADPH-cytochrome c reductase activity of both proteins, Nitric Oxide 1 (1997) 39 – 49. [60] P. Lane, S.S. Gross, The autoinhibitory control element and calmodulin conspire to provide physiological modulation of endothelial and neuronal nitric oxide synthase activity, Acta Physiol. Scand. 168 (2000) 53 – 63. [61] L.J. Roman, P. Marta´sek, B.S.S. Masters, Intrinsic and extrinsic modulation of nitric oxide synthase activity, Chem. Rev. 102 (2002) 1179 – 1189. [62] A.P. Ledbetter, K. McMillan, L.J. Roman, B.S.S. Masters, J.H. Dawson, M. Sono, Low-temperature stabilization and spectroscopic characterization of the dioxygen complex of the ferrous neuronal nitric oxide synthase oxygenase domain, Biochemistry 38 (1999) 8014 – 8021. [63] H.M. Abu-Soud, J. Wang, D.L. Rousseau, J.M. Fukuto, L.J. Ignarro, D.J. Stuehr, Neuronal nitric oxide synthase self-inactivates by forming a ferrous-nitrosyl complex during aerobic catalysis, J. Biol. Chem. 270 (1995) 22997 – 23006. [64] J. Santolini, S. Adak, C.M.L. Curran, D.J. Stuehr, A kinetic simulation model that describes catalysis and regulation in nitric-oxide synthase, J. Biol. Chem. 276 (2001) 1233 – 1243.