Substrate-ligand interactions in Geobacillus stearothermophilus nitric oxide synthase

Nitric oxide synthase (NOS) generates NO via a sequential two-step reaction [l-arginine (l-Arg) --> N-hydroxy-l-arginine (NOHA) --> l-citrulline + NO]. Each step of the reaction follows a distinct mechanism defined by the chemical environment introduced by each substrate bound to the heme active site. The dioxygen complex of the NOS enzyme from a thermophilic bacterium, Geobacillus stearothermophilus (gsNOS), is unusually stable; hence, it provides a unique model for the studies of the mechanistic differences between the two steps of the NOS reaction. By using CO as a structural probe, we found that gsNOS exhibits two conformations in the absence of substrate, as indicated by the presence of two sets of nu(Fe-CO)/nu(C-O) modes in the resonance Raman spectra. In the nu(Fe-CO) versus nu(C-O) inverse correlation plot, one set of data falls on the correlation line characterized by mammalian NOSs (mNOS), whereas the other set of data lies on a new correlation line defined by a bacterial NOS from Bacillus subtilis (bsNOS), reflecting a difference in the proximal Fe-Cys bond strength in the two conformers of gsNOS. The addition of l-Arg stabilizes the conformer associated with the mNOS correlation line, whereas NOHA stabilizes the conformer associated with the bsNOS correlation line, although both substrates introduce a positive electrostatic potential into the distal heme pocket. To assess how substrate binding affects Fe-Cys bond strength, the frequency of the Fe-Cys stretching mode of gsNOS was monitored by resonance Raman spectroscopy with 363.8 nm excitation. In the substrate-free form, the Fe-Cys stretching mode was detected at 342.5 cm(-1), similar to that of bsNOS. The binding of l-Arg and NOHA brings about a small decrease and increase in the Fe-Cys stretching frequency, respectively. The implication of these unique structural features with respect to the oxygen chemistry of NOS is discussed.     

Nitrosyl-heme structures of Bacillus subtilis nitric oxide synthase have implications for understanding substrate oxidation

The crystal structures of nitrosyl-heme complexes of a prokaryotic nitric oxide synthase (NOS) from Bacillus subtilis (bsNOS) reveal changes in active-site hydrogen bonding in the presence of the intermediate N(omega)-hydroxy-l-arginine (NOHA) compared to the substrate l-arginine (l-Arg). Correlating with a Val-to-Ile residue substitution in the bsNOS heme pocket, the Fe(II)-NO complex with both l-Arg and NOHA is more bent than the Fe(II)-NO, l-Arg complex of mammalian eNOS [Li, H., Raman, C. S., Martasek, P., Masters, B. S. S., and Poulos, T. L. (2001) Biochemistry 40, 5399-5406]. Structures of the Fe(III)-NO complex with NOHA show a nearly linear nitrosyl group, and in one subunit, partial nitrosation of bound NOHA. In the Fe(II)-NO complexes, the protonated NOHA N(omega) atom forms a short hydrogen bond with the heme-coordinated NO nitrogen, but active-site water molecules are out of hydrogen bonding range with the distal NO oxygen. In contrast, the l-Arg guanidinium interacts more weakly and equally with both NO atoms, and an active-site water molecule hydrogen bonds to the distal NO oxygen. This difference in hydrogen bonding to the nitrosyl group by the two substrates indicates that interactions provided by NOHA may preferentially stabilize an electrophilic peroxo-heme intermediate in the second step of NOS catalysis.     

Single-turnover of nitric-oxide synthase in the presence of 4-amino-tetrahydrobiopterin: proposed role for tetrahydrobiopterin as a proton donor

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric-oxide synthase (NOS) that serves as a one-electron donor to the oxyferrous.heme complex. 4-Aminotetrahydrobiopterin (4-amino-BH4) is a potent inhibitor of NO synthesis, although it mimics all allosteric and structural effects of BH4 and exhibits comparable redox properties. We studied the reaction of reduced endothelial NOS oxygenase domain with O2 in the presence of 4-amino-BH4 at -30 degrees C by optical and electron paramagnetic resonance (EPR) spectroscopy. With Arg as the substrate, we observed a trihydropteridine radical with a corresponding heme species that was oxyferrous, with a Soret maximum at 428 nm and no EPR signal. With NG-hydroxy-l-arginine (NHA) no pterin radical appeared, whereas an axial ferrous heme.NO complex was formed. The corresponding optical spectra, with Soret bands at 417/423 nm, suggest that the proximal sulfur ligand is protonated. Accordingly, 4-amino-BH4 serves as a one-electron donor to Fe(II).O2 with both Arg and NHA, but the reaction cycle cannot be completed with either substrate. We propose that protonation of Fe(II)O2- is inhibited in the presence of 4-amino-BH4. With Arg, dissociation of O2- and binding of O2 yields Fe(II).O2 and a pteridine radical; with NHA, reaction of the substrate with heme-bound O2- eventually yields Fe(II).NO and reduced 4-amino-BH4. These results suggest that BH4 donates a proton to Fe(II).O2- during catalysis and that inhibition by 4-amino-BH4 may be due to its inability to support this essential protonation step.     

Electron transfer, oxygen binding, and nitric oxide feedback inhibition in endothelial nitric-oxide synthase

We studied steps that make up the initial and steady-state phases of nitric oxide (NO) synthesis to understand how activity of bovine endothelial NO synthase (eNOS) is regulated. Stopped-flow analysis of NADPH-dependent flavin reduction showed the rate increased from 0. 13 to 86 s(-1) upon calmodulin binding, but this supported slow heme reduction in the presence of either Arg or N(omega)-hydroxy-l-arginine (0.005 and 0.014 s(-1), respectively, at 10 degrees C). O(2) binding to ferrous eNOS generated a transient ferrous dioxy species (Soret peak at 427 nm) whose formation and decay kinetics indicate it can participate in NO synthesis. The kinetics of heme-NO complex formation were characterized under anaerobic conditions and during the initial phase of NO synthesis. During catalysis heme-NO complex formation required buildup of relatively high solution NO concentrations (>50 nm), which were easily achieved with N(omega)-hydroxy-l-arginine but not with Arg as substrate. Heme-NO complex formation caused eNOS NADPH oxidation and citrulline synthesis to decrease 3-fold and the apparent K(m) for O(2) to increase 6-fold. Our main conclusions are: 1) The slow steady-state rate of NO synthesis by eNOS is primarily because of slow electron transfer from its reductase domain to the heme, rather than heme-NO complex formation or other aspects of catalysis. 2) eNOS forms relatively little heme-NO complex during NO synthesis from Arg, implying NO feedback inhibition has a minimal role. These properties distinguish eNOS from the other NOS isoforms and provide a foundation to better understand its role in physiology and pathology.     

Crystal structures of CO and NO adducts of MauG in complex with pre-methylamine dehydrogenase: implications for the mechanism of dioxygen activation

MauG is a diheme enzyme responsible for the post-translational formation of the catalytic tryptophan tryptophylquinone (TTQ) cofactor in methylamine dehydrogenase (MADH). MauG can utilize hydrogen peroxide, or molecular oxygen and reducing equivalents, to complete this reaction via a catalytic bis-Fe(IV) intermediate. Crystal structures of diferrous, Fe(II)-CO, and Fe(II)-NO forms of MauG in complex with its preMADH substrate have been determined and compared to one another as well as to the structure of the resting diferric MauG-preMADH complex. CO and NO each bind exclusively to the 5-coordinate high-spin heme with no change in ligation of the 6-coordinate low-spin heme. These structures reveal likely roles for amino acid residues in the distal pocket of the high-spin heme in oxygen binding and activation. Glu113 is implicated in the protonation of heme-bound diatomic oxygen intermediates in promoting cleavage of the O-O bond. Pro107 is shown to change conformation on the binding of each ligand and may play a steric role in oxygen activation by positioning the distal oxygen near Glu113. Gln103 is in a position to provide a hydrogen bond to the Fe(IV)═O moiety that may account for the unusual stability of this species in MauG.     

Formation and reactions of the heme-dioxygen intermediate in the first and second steps of nitric oxide synthesis as studied by stopped-flow spectroscopy under single-turnover conditions

To better understand the mechanism of nitric oxide (NO) synthesis, we studied conversion of N-hydroxy-L-arginine (NOHA) or L-arginine (Arg) to citrulline and NO under single-turnover conditions using the oxygenase domain of neuronal nitric oxide synthase (nNOSoxy) and rapid scanning stopped-flow spectroscopy. When anaerobic nNOSoxy saturated with H(4)B and NOHA was provided with 0.5 or 1 electron per heme and then exposed to air at 25 degrees C, it formed 0.5 or 1 mol of citrulline/mol of heme, respectively, indicating that NOHA conversion had 1:1 stoichiometry with respect to electrons added. Identical experiments with Arg produced substoichiometric amounts of NOHA or citrulline even when up to 3 electrons were provided per heme. Transient spectral intermediates were investigated at 10 degrees C. For NOHA, four species were observed in the following sequence: starting ferrous nNOSoxy, a transient ferrous-dioxygen complex, a transient ferric-NO complex, and ferric nNOSoxy. For Arg, transient intermediates other than the ferrous-dioxygen species were not apparent during the reaction. Our results provide a kinetic framework for formation and reactions of the ferrous-dioxygen complex in each step of NO synthesis and establish that (1) the ferrous-dioxy enzyme reacts quantitatively with NOHA but not with Arg and (2) its reaction with NOHA forms 1 NO/heme, which immediately binds to form a ferric heme-NO complex.     

Heme insertion, assembly, and activation of apo-neuronal nitric-oxide synthase in vitro

It has been established that in the case of inducible NO synthase (NOS), a functionally active homodimer is assembled from the heme-deficient monomeric apo-NOS in vitro by the addition of heme, whereas the heme-deficient neuronal isoform (apo-nNOS) is at best only partially activated. In the current study we have discovered that reactive oxygen species, which can be removed by the addition of superoxide dismutase and catalase, destroy the heme and limit the activation of apo-nNOS in vitro. With the use of these improved conditions, we show for the first time that heme insertion is a rapid process that results in formation of a heme-bound monomeric nNOS that is able to form the ferrous-CO P450 complex but is unable to synthesize NO. A slow process requiring more than 90 min is required for dimerization and activation of this P450 intermediate to give an enzyme with a specific activity of approximately 1100 nmol of NO formed/min/mg of protein, similar to that of the native enzyme. Interestingly, the dimer is not SDS-resistant and is not the same dimer that forms in vivo. These studies indicate at least two intermediates in the assembly of nNOS and advance our understanding of the regulation of nNOS.     

A conserved Val to Ile switch near the heme pocket of animal and bacterial nitric-oxide synthases helps determine their distinct catalytic profiles

Nitric oxide (NO) release from nitric oxide synthases (NOSs) is largely dependent on the dissociation of an enzyme ferric heme-NO product complex (Fe(III)NO). Although the NOS-like protein from Bacillus subtilis (bsNOS) generates Fe(III)NO from the reaction intermediate N-hydroxy-l-arginine (NOHA), its NO dissociation is about 20-fold slower than in mammalian NOSs. Crystal structures suggest that a conserved Val to Ile switch near the heme pocket of bsNOS might determine its kinetic profile. To test this we generated complementary mutations in the mouse inducible NOS oxygenase domain (iNOSoxy, V346I) and in bsNOS (I224V) and characterized the kinetics and extent of their NO synthesis from NOHA and their NO-binding kinetics. The mutations did not greatly alter binding of Arg, (6R)-tetrahydrobiopterin, or alter the electronic properties of the heme or various heme-ligand complexes. Stopped-flow spectroscopy was used to study heme transitions during single turnover NOHA reactions. I224V bsNOS displayed three heme transitions involving four species as typically occurs in wild-type NOS, the beginning ferrous enzyme, a ferrous-dioxy (Fe(II)O(2)) intermediate, Fe(III)NO, and an ending ferric enzyme. The rate of each transition was increased relative to wild-type bsNOS, with Fe(III)NO dissociation being 3.6 times faster. In V346I iNOSoxy we consecutively observed the beginning ferrous, Fe(II)O(2), a mixture of Fe(III)NO and ferric heme species, and ending ferric enzyme. The rate of each transition was decreased relative to wild-type iNOSoxy, with the Fe(III)NO dissociation being 3 times slower. An independent measure of NO binding kinetics confirmed that V346I iNOSoxy has slower NO binding and dissociation than wild-type. Citrulline production by both mutants was only slightly lower than wild-type enzymes, indicating good coupling. Our data suggest that a greater shielding of the heme pocket caused by the Val/Ile switch slows down NO synthesis and NO release in NOS, and thus identifies a structural basis for regulating these kinetic variables.     

Influence of heme-thiolate in shaping the catalytic properties of a bacterial nitric-oxide synthase

Nitric-oxide synthases (NOS) are heme-thiolate enzymes that generate nitric oxide (NO) from L-arginine. Mammalian and bacterial NOSs contain a conserved tryptophan (Trp) that hydrogen bonds with the heme-thiolate ligand. We mutated Trp(66) to His and Phe (W66H, W66F) in B. subtilis NOS to investigate how heme-thiolate electronic properties control enzyme catalysis. The mutations had opposite effects on heme midpoint potential (-302, -361, and -427 mV for W66H, wild-type (WT), and W66F, respectively). These changes were associated with rank order (W66H < WT < W66F) changes in the rates of oxygen activation and product formation in Arg hydroxylation and N-hydroxyarginine (NOHA) oxidation single turnover reactions, and in the O(2) reactivity of the ferrous heme-NO product complex. However, enzyme ferrous heme-O(2) autoxidation showed an opposite rank order. Tetrahydrofolate supported NO synthesis by WT and the mutant NOS. All three proteins showed similar extents of product formation (L-Arg → NOHA or NOHA → citrulline) in single turnover studies, but the W66F mutant showed a 2.5 times lower activity when the reactions were supported by flavoproteins and NADPH. We conclude that Trp(66) controls several catalytic parameters by tuning the electron density of the heme-thiolate bond. A greater electron density (as in W66F) improves oxygen activation and reactivity toward substrate, but decreases heme-dioxy stability and lowers the driving force for heme reduction. In the WT enzyme the Trp(66) residue balances these opposing effects for optimal catalysis.     

Low-temperature optical absorption spectra suggest a redox role for tetrahydrobiopterin in both steps of nitric oxide synthase catalysis

To investigate the role of tetrahydrobiopterin (BH4) in the catalytic mechanism of nitric oxide synthase (NOS), we analyzed the spectral changes following addition of oxygen to the reduced oxygenase domain of endothelial nitric oxide synthase (NOS) in the presence of different pteridines at -30 degrees C. In the presence of N(G)-hydroxy-L-arginine (NOHLA) and BH4 or 5-methyl-BH4, both of which support NO synthesis, the first observable species were mixtures of high-spin ferric NOS (395 nm), ferric NO-heme (439 nm), and the oxyferrous complex (417 nm). With Arg, no clear intermediates could be observed under the same conditions. In the presence of the BH4-competitive inhibitor 7,8-dihydrobiopterin (BH2), intermediates with maxima at 417 and 425 nm were formed in the presence of Arg and NOHLA, respectively. In the presence of 4-amino-BH4, the maxima of the intermediates with Arg and NOHLA were at 431 and 423 nm, respectively. We ascribe all four spectra to oxyferrous heme complexes. The intermediates observed in this study slowly decayed to the high-spin ferric state at -30 degrees C, except for those formed in the presence of 4-amino-BH4, which required warming to room temperature for regeneration of high-spin ferric NOS; with Arg, regeneration remained incomplete. From these observations, we draw several conclusions. (1) BH4 is required for reductive oxygen activation, probably as a transient one-electron donor, not only in the reaction with Arg but also with NOHLA; (2) in the absence of redox-active pterins, reductive oxygen activation does not occur, which results in accumulation of the oxyferrous complex; (3) the spectral properties of the oxyferrous complex are affected by the presence and identity of the substrate; (4) the slow and incomplete formation of high-spin ferric heme with 4-amino-BH4 suggests a structural cause for inhibition of NOS activity by this pteridine.     

Effects of the new arginase inhibitor N(omega)-hydroxy-nor-L-arginine on NO synthase activity in murine macrophages.

In stimulated murine macrophage, arginase and nitric oxide synthase (NOS) compete for their common substrate, l-arginine. The objectives of this study were (i) to test the new alpha-amino acid N(omega)-hydroxy-nor-l-arginine (nor-NOHA) as a new selective arginase inhibitor and (ii) to elucidate the effects of arginase inhibition on l-arginine utilization by an inducible NOS. Nor-NOHA is about 40-fold more potent than N(omega)-hydroxy-l-arginine (NOHA), an intermediate in the l-arginine/NO pathway, to inhibit the hydrolysis of l-arginine to l-ornithine catalyzed by unstimulated murine macrophages (IC(50) values 12 +/- 5 and 400 +/- 50 microM, respectively). Stimulation of murine macrophages with interferon-gamma and lipopolysaccharide (IFN-gamma + LPS) results in clear expression of an inducible NOS (iNOS) and to an increase in arginase activity. Nor-NOHA is also a potent inhibitor of arginase in IFN-gamma + LPS-stimulated macrophage (IC(50) value 10 +/- 3 microM). In contrast to NOHA, nor-NOHA is neither a substrate nor an inhibitor for iNOS and it appears as a useful tool to study the interplays between arginase and NOS. Inhibition of arginase by nor-NOHA increases nitrite and l-citrulline accumulation for incubation times higher than 12 h, under our conditions. Our results allow the determination of the kinetic parameters of the two competitive pathways and the proposal of a simple model which readily explains the differences observed between experiments. This model readily accounts for the observed effects and should be useful to predict the consequences of arginase inhibition in the presence of an active NOS on l-arginine availability.     

Revisiting heme mechanisms. A perspective on the mechanisms of nitric oxide synthase (NOS), Heme oxygenase (HO), and cytochrome P450s (CYP450s)

Despite the essential biological importance of reactions that involve heme, mechanisms of heme reactions in enzymes like nitric oxide synthase (NOS), heme oxygenase (HO), and cytochrome P450s (CYP450s) are still not well-understood. This Perspective on NOS, HO, and CYP450 mechanisms is written from the point of view of the heme chemistry. Steps in the classical heme catalytic cycle are discussed based on the specific environment within each of these enzymes. Elucidation of the mechanisms of NOS inactivation by some substrate analogues provides important mechanistic clues to the NOS catalytic mechanism. On the basis of mechanistic studies of NOS inactivation by amidine analogues of l-arginine and other previous mechanistic results, a new mechanism for NOS-catalyzed l-arginine NG-hydroxylation (the first half of the catalytic reaction) is proposed in this Perspective. The key step in the second half of the NOS catalytic reaction, the internal electron transfer between the substrate and heme, is discussed on the basis of mechanistic results of NOS inactivation by NG-allyl-l-arginine and the structures of the substrate intermediates. Elucidation of the mechanism of NOS inactivation by amidines, which leads to heme degradation, also provides important mechanistic implications for heme oxygenase-catalyzed heme catabolism. Focusing on the meso-hydroxylation step during inactivation of NOS by amidines as well as the HO-catalyzed reaction, the essential nature of the heme-oxygen species responsible for porphyrin meso-hydroxylation is discussed. Finally, on the basis of the proposed heme degradation mechanism during NOS inactivation and the HO-catalyzed reaction, the mechanism for the formation of the monooxygenated heme species in P450-catalyzed reactions is discussed.     

NO binding induced conformational changes in a truncated hemoglobin from Mycobacterium tuberculosis

The resonance Raman spectra of the NO-bound ferric derivatives of wild-type HbN and the B10 Tyr --> Phe mutant of HbN, a hemoglobin from Mycobacterium tuberculosis, were examined with both Soret and UV excitation. The Fe-N-O stretching and bending modes of the NO derivative of the wild-type protein were tentatively assigned at 591 and 579 cm(-1), respectively. Upon B10 mutation, the Fe-NO stretching mode was slightly enhanced and the bending mode diminished in amplitude. In addition, the N-O stretching mode shifted from 1914 to 1908 cm(-1). These data suggest that the B10 Tyr forms an H-bond(s) with the heme-bound NO and causes it to bend in the wild-type protein. To further investigate the interaction between the B10 Tyr and the heme-bound NO, we examined the UV Raman spectrum of the B10 Tyr by subtracting the B10 mutant spectrum from the wild-type spectrum. It was found that, upon NO binding to the ferric protein, the Y(8a) mode of the B10 Tyr shifted from 1616 to 1622 cm(-1), confirming a direct interaction between the B10 Tyr and the heme-bound NO. Furthermore, the Y(8a) mode of the other two Tyr residues at positions 16 and 72 that are remote from the heme was also affected by NO binding, suggesting that NO binding to the distal site of the heme triggers a large-scale conformational change that propagates through the pre-F helix loop to the E and B helices. This large-scale conformational change triggered by NO binding may play an important role in regulating the ligand binding properties and/or the chemical reactivity of HbN.     

Geminate recombination of nitric oxide to endothelial nitric-oxide synthase and mechanistic implications.

The nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to L-citrulline and NO through consumption of oxygen bound to the heme. Because NO is produced close to the heme and may bind to it, its subsequent role in a regulatory mechanism should be scrutinized. We therefore examined the kinetics of NO rebinding after photodissociation in the heme pocket of human endothelial NOS by means of time-resolved absorption spectroscopy. We show that geminate recombination of NO indeed occurs and that this process is strongly modulated by L-Arg. This NO rebinding occurs in a multiphasic fashion and spans over 3 orders of magnitude. In both ferric and ferrous states of the heme, a fast nonexponential picosecond geminate rebinding first takes place followed by a slower nanosecond phase. The rates of both phases decreased, whereas their relative amplitudes are changed by the presence of L-Arg; the overall effect is a slow down of NO rebinding. For the isolated oxygenase domain, the picosecond rate is unchanged, but the relative amplitude of the nanosecond binding decreased. We assigned the nanosecond kinetic component to the rebinding of NO that is still located in the protein core but not in the heme pocket. The implications for a mechanism of regulation involving NO binding are discussed.     

Oxygenase domain of Drosophila melanogaster nitric oxide synthase: unique kinetic parameters enable a more efficient NO release

Although nitric oxide (NO) is important for cell signaling and nonspecific immunity in the fruit fly Drosophila melanogaster, little is known about its single NO synthase (dNOS). We expressed the oxygenase domain of dNOS (dNOSoxy), characterized its spectroscopic, kinetic, and catalytic properties, and interpreted them in light of a global kinetic model for NO synthesis. Single turnover reactions with ferrous dNOSoxy showed it could convert Arg to N'omega-hydroxy-l-arginine (NOHA), or NOHA to citrulline and NO, when it was given 6R-tetrahydrobiopterin and O2. The dNOSoxy catalyzed Arg hydroxylation and NOHA oxidation at rates that matched or exceeded the rates catalyzed by the three mammalian NOSoxy enzymes. Consecutive heme-dioxy, ferric heme-NO, and ferric heme species were observed in the NOHA reaction of dNOSoxy, indicating that its catalytic mechanism is the same as in the mammalian NOS. However, NO dissociation from dNOSoxy was 4 to 9 times faster than that from the mammalian NOS enzymes. In contrast, the dNOSoxy ferrous heme-NO complex was relatively unreactive toward O2 and in this way was equivalent to the mammalian neuronal NOS. Our data show that dNOSoxy has unique settings for the kinetic parameters that determine its NO synthesis. Computer simulations reveal that these unique settings should enable dNOS to be a more efficient and active NO synthase than the mammalian NOS enzymes, which may allow it to function more broadly in cell signaling and immune functions in the fruit fly.     

Oxygen-induced radical intermediates in the nNOS oxygenase domain regulated by L-arginine, tetrahydrobiopterin, and thiol

Fully coupled nitric oxide synthase (NOS) catalyzes formation of nitric oxide (NO), l-citrulline, NADP+, and water from l-arginine, NADPH, and oxygen. Uncoupled or partially coupled NOS catalyzes the synthesis of reactive oxygen species such as superoxide, hydrogen peroxide, and peroxynitrite, depending on the availability of cofactor tetrahydrobiopterin (BH4) and l-arginine during catalysis. We identified three distinct oxygen-induced radical intermediates in the ferrous endothelial NOS oxygenase domain (eNOSox) with or without BH4 and/or l-arginine [Berka, V., Wu, G., Yeh, H. C., Palmer, G., and Tsai, A.-L. (2004) J. Biol. Chem. 279, 32243-32251]. The effects of BH4 and l-arginine on the oxygen-induced radical intermediates in the isolated neuronal NOS oxygenase domain (nNOSox) have been similarly investigated by single-turnover stopped-flow and rapid-freeze quench EPR kinetic measurements in the presence or absence of dithiothreitol (DTT). Like for eNOSox, we found different radical intermediates in the reaction of ferrous nNOSox with oxygen. (1) nNOSox (without BH4 or l-Arg) produces superoxide in the presence or absence of DTT. (2) nNOSox (with BH4 and l-Arg) yields a typical BH4 radical in a manner independent of DTT. (3) nNOSox (with BH4 and without l-Arg) yields a new radical. Without DTT, EPR showed a mixture of superoxide and biopterin radicals. With DTT, a new approximately 75 G wide radical EPR was observed, different from the radical formed by eNOSox. (4) The presence of only l-arginine in nNOSox (without BH4 but with l-Arg) caused conversion of approximately 70% of superoxide radical to a novel radical, explaining how l-arginine decreases the level of superoxide production in nNOSox (without BH4 but with l-Arg). The regulatory role of l-arginine in nNOS is thus very different from that in eNOS where substrate was only to decrease the rate of formation of superoxide but not the total amount of radical. The role of DTT is also different. DTT prevents oxidation of BH4 in both isoforms, but in nNOS, DTT also inhibits oxidation of two key cysteines in nNOSox to prevent the loss of substrate binding. This new role of thiol found only for nNOS may be significant in neurodegenerative diseases.     

EPR and ENDOR characterization of intermediates in the cryoreduced oxy-nitric oxide synthase heme domain with bound L-arginine or N(G)-hydroxyarginine

Reconstitution of the endothelial nitric oxide synthase heme domain (NOS) with the catalytically noncompetent 4-aminotetrahydrobiopterin has allowed us to prepare at -40 degrees C the oxyferrous-NOS-substrate complexes of both L-arginine (Arg) and N(G)-hydroxyarginine (NOHA). We have radiolytically cryoreduced these complexes at 77 K and used EPR and ENDOR spectroscopies to characterize the initial products of reduction, as well as intermediates that arise during stepwise annealing to higher temperatures. Peroxo-ferri-NOS is the primary product of 77 K cryoreduction when either Arg or NOHA is the substrate. Proton ENDOR spectra of this state suggest that the peroxo group is H-bonded to a [guanidinium-water] network that forms because the binding of O2 to the ferroheme of NOS recruits H2O. At no stage of reaction/annealing does one observe an EPR signal from a hydroperoxo-ferri state with either substrate. Instead, peroxo-ferri-NOS-substrate complexes convert to a product-state intermediate at the extremely low temperature of 165-170 K. EPR and proton ENDOR spectra of the intermediate formed with Arg as substrate support the suggestion that the reaction involves the formation and attack of Compound I. Within the time/temperature resolution of the present experiments, samples with Arg and NOHA as substrate behave the same in the initial steps of cryoreduction/annealing, despite the different acid/base characteristics of the two substrates. This leads us to discuss the possibility that ambient-temperature catalytic conversion of both substrates is initiated by reduction of the oxy-ferroheme to the hydroperoxo-ferriheme through a coupled proton-electron transfer from a heme-pocket reductant, and that Arg may provide the stoichiometrically second proton of catalysis.     

Magnetic circular dichroism spectroscopic characterization of the NOS-like protein from Geobacillus stearothermophilus (gsNOS)

Nitric oxide synthase (NOS) catalyzes the NADPH- and O₂-dependent oxidation of l-arginine (l-Arg) to nitric oxide (NO) and citrulline via an N^G-hydroxy-l-arginine (NHA) intermediate. Mammalian NOSs have been studied quite extensively; other eukaryotes and some prokaryotes appear to express NOS-like proteins comparable to the oxygenase domain of mammalian NOSs. In this study, a recombinant NOS-like protein from the thermostable bacterium Geobacillus stearothermophilus (gsNOS) has been characterized using magnetic circular dichroism (MCD) and UV–Vis absorption spectroscopic techniques. Spectral comparisons of ligand complexes (with O₂, NO and CO) of substrate-bound (l-Arg or NHA) gsNOS, including the key oxyferrous complex studied at ?50 °C in cryogenic mixed solvents, with analogous mammalian NOS complexes indicate overall spectroscopic similarities between gsNOS and mammalian NOSs. However, more detailed spectral comparisons reflect subtle structural differences between gsNOS and mammalian NOSs. This may be due to an incomplete tetrahydrobiopterin (BH₄)-binding site and low BH₄-binding affinity, which may become even lower in the presence of cryosolvent in gsNOS. Although BH₄-binding may be altered, gsNOS appears to require the pterin for NO production since formation of the stable ferric-NO product complex was only observed when excess BH₄ (>150 μM) over gsNOS was present upon single turnover reaction in which O₂ was bubbled into dithionite-reduced NHA-bound protein solution at ?35 °C or ?50 °C.     

Ligand-protein interactions in nitric oxide synthase

Nitric oxide synthases (NOSs) are heme proteins that catalyze the formation of nitric oxide (NO) from L-arginine and oxygen in a sequential two-step process. Three structurally similar isoforms have been identified that deliver NO to different tissues for specific functions. An understanding of the interactions of ligands with the protein is essential to determine the mechanism of catalysis, the design of inhibitors and the differential auto-inhibitory regulation of the enzymatic activity of the isoforms due to the binding of NO to the heme. Ligand-protein interactions in the three isoforms revealed by resonance Raman scattering studies are reviewed in this article. The CO-related modes in the CO-bound ferrous enzyme are sensitive to the presence of substrate, either L-arginine or N-hydroxy-L-arginine, in the distal pocket, but insensitive to the presence of the tetrahydrobiopterin (H4B) cofactor. In contrast, when NO is coordinated to the ferric heme, the NO is sensitive to the substrate only when H4B is present. Furthermore, in the NO-bound ferric enzyme, the addition of H4B induces a large heme distortion that may modulate heme reduction and thereby regulate the NO auto-inhibitory process. In the metastable O2-bound enzyme, L-arginine binding causes the appearance of a shoulder on the O-O stretching mode, suggesting a specific interaction of the heme-bound dioxygen with the bound-substrate that may be crucial for the oxygenation reaction of the substrate during the catalytic turn-over. It is postulated that spectroscopic differences in the oxy-complex are a consequence of the degree of protonation of the proximal cysteine ligand on the heme. Resonance Raman studies of NOSs expand our understanding of the mechanistic features of this important family of enzymes.     

Trp180 of endothelial NOS and Trp56 of bacterial saNOS modulate sigma bonding of the axial cysteine to the heme

The proximal ligand of thiolate-coordinated heme proteins is crucial for the activation of the oxygen molecule and hydroxylation of substrates. In nitric oxide synthases (NOSs), the heme axial cysteine ligand forms a hydrogen bond to the side chain indole nitrogen of a tryptophan residue. Resonance Raman spectroscopy was used to probe W56F and W56Y variants of the NOS of Staphylococcus aureus (saNOS) and the analogous W180 variants of the endothelial NOS oxygenase domain (eNOSox). We show that the variants displayed lower ν_Fe-NO and ν_Fe-CO frequencies indicating that these mutations increased the electron density on the axial cysteine in their Fe^IIINO and Fe^IICO complexes. We also show by UV-visible spectroscopy that the Fe^IICO complexes of the variants displayed a red-shifted Soret optical transition in addition to the lower ν_Fe-CO thus establishing that these properties are sensitive indicators of the modulation of the basicity of the axial cysteine. We infer, based on its spectroscopic properties, that ferrous eNOSox W180Y saturated with l-arginine and tetrahydrobiopterin forms a tyrosine-cysteine hydrogen bond when bound to CO. Evidence for such a hydrogen bond was not obtained for the Fe^IIINO protein nor for the analogous saNOS variant. These mutations reveal interesting differences in the response of NOS isotypes to analogous mutations at conserved residues and clearly show that the heme-Fe to cysteine σ bond is modulated by the Cys-Trp hydrogen bond in NOSs. These studies serve as a basis to gain information on the role played by this hydrogen bond in oxygen activation in this class of enzymes.