Reductase domain of Drosophila melanogaster nitric-oxide synthase: redox transformations, regulation, and similarity to mammalian homologues

The nitric oxide synthase of Drosophila melanogaster (dNOS) participates in essential developmental and behavioral aspects of the fruit fly, but little is known about dNOS catalysis and regulation. To address this, we expressed a construct comprising the dNOS reductase domain and its adjacent calmodulin (CaM) binding site (dNOSr) and characterized the protein regarding its catalytic, kinetic, and regulatory properties. The Ca2+ concentration required for CaM binding to dNOSr was between that of the mammalian endothelial and neuronal NOS enzymes. CaM binding caused the cytochrome c reductase activity of dNOSr to increase 4 times and achieve an activity comparable to that of mammalian neuronal NOS. This change was associated with decreased shielding of the FMN cofactor from solvent and an increase in the rate of NADPH-dependent flavin reduction. Flavin reduction in dNOSr was relatively slow following the initial 2-electron reduction, suggesting a slow inter-flavin electron transfer, and no charge-transfer complex was observed between bound NADP+ and reduced FAD during the process. We conclude that dNOSr catalysis and regulation is most similar to the mammalian neuronal NOS reductase domain, although differences exist in their flavin reduction behaviors. The apparent conservation between the fruit fly and mammalian enzymes is consistent with dNOS operating in various signal cascades that involve NO.     

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.     

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.     

A tetrahydrobiopterin radical forms and then becomes reduced during Nomega-hydroxyarginine oxidation by nitric-oxide synthase

Nitric-oxide synthases are flavoheme enzymes that catalyze two sequential monooxygenase reactions to generate nitric oxide (NO) from l-arginine. We investigated a possible redox role for the enzyme-bound cofactor 6R-tetrahydrobiopterin (H4B) in the second reaction of NO synthesis, which is conversion of N-hydroxy-l-arginine (NOHA) to NO plus citrulline. We used stopped-flow spectroscopy and rapid-freeze EPR spectroscopy to follow heme and biopterin transformations during single-turnover NOHA oxidation reactions catalyzed by the oxygenase domain of inducible nitric-oxide synthase (iNOSoxy). Significant biopterin radical (>0.5 per heme) formed during reactions catalyzed by iNOSoxy that contained either H4B or 5-methyl-H4B. Biopterin radical formation was kinetically linked to conversion of a heme-dioxy intermediate to a heme-NO product complex. The biopterin radical then decayed within a 200-300-ms time period just prior to dissociation of NO from a ferric heme-NO product complex. Measures of final biopterin redox status showed that biopterin radical decay occurred via an enzymatic one-electron reduction process that regenerated H4B (or 5MeH4B). These results provide evidence of a dual redox function for biopterin during the NOHA oxidation reaction. The data suggest that H4B first provides an electron to a heme-dioxy intermediate, and then the H4B radical receives an electron from a downstream reaction intermediate to regenerate H4B. The first one-electron transition enables formation of the heme-based oxidant that reacts with NOHA, while the second one-electron transition is linked to formation of a ferric heme-NO product complex that can release NO from the enzyme. These redox roles are novel and expand our understanding of biopterin function in biology.     

Arginine conversion to nitroxide by tetrahydrobiopterin-free neuronal nitric-oxide synthase. Implications for mechanism

We studied catalysis by tetrahydrobiopterin (H4B)-free neuronal nitric-oxide synthase (nNOS) to understand how heme and H4B participate in nitric oxide (NO) synthesis. H4B-free nNOS catalyzed Arg oxidation to N(omega)-hydroxy-l-Arg (NOHA) and citrulline in both NADPH- and H(2)O(2)-driven reactions. Citrulline formation was time- and enzyme concentration-dependent but was uncoupled relative to NADPH oxidation, and generated nitrite and nitrate without forming NO. Similar results were observed when NOHA served as substrate. Steady-state and stopped-flow spectroscopy with the H4B-free enzyme revealed that a ferrous heme-NO complex built up after initiating catalysis in both NADPH- and H(2)O(2)-driven reactions, consistent with formation of nitroxyl as an immediate product. This differed from the H4B-replete enzyme, which formed a ferric heme-NO complex as an immediate product that could then release NO. We make the following conclusions. 1) H4B is not essential for Arg oxidation by nNOS, although it helps couple NADPH oxidation to product formation in both steps of NO synthesis. Thus, the NADPH- or H(2)O(2)-driven reactions form common heme-oxy species that can react with substrate in the presence or absence of H4B. 2) The sole essential role of H4B is to enable nNOS to generate NO instead of nitroxyl. On this basis we propose a new unified model for heme-dependent oxygen activation and H4B function in both steps of NO synthesis.     

A kinetic simulation model that describes catalysis and regulation in nitric-oxide synthase

After initiating NO synthesis a majority of neuronal NO synthase (nNOS) quickly partitions into a ferrous heme-NO complex. This down-regulates activity and increases enzyme K(m,O(2)). To understand this process, we developed a 10-step kinetic model in which the ferric heme-NO enzyme forms as the immediate product of catalysis, and then partitions between NO dissociation versus reduction to a ferrous heme-NO complex. Rate constants used for the model were derived from recent literature or were determined here. Computer simulations of the model precisely described both pre-steady and steady-state features of nNOS catalysis, including NADPH consumption and NO production, buildup of a heme-NO complex, changes between pre-steady and steady-state rates, and the change in enzyme K(m,O(2)) in the presence or absence of NO synthesis. The model also correctly simulated the catalytic features of nNOS mutants W409F and W409Y, which are hyperactive and display less heme-NO complex formation in the steady state. Model simulations showed how the rate of heme reduction influences several features of nNOS catalysis, including populations of NO-bound versus NO-free enzyme in the steady state and the rate of NO synthesis. The simulation predicts that there is an optimum rate of heme reduction that is close to the measured rate in nNOS. Ratio between NADPH consumption and NO synthesis is also predicted to increase with faster heme reduction. Our kinetic model is an accurate and versatile tool for understanding catalytic behavior and will provide new perspectives on NOS regulation.     

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.     

A tryptophan that modulates tetrahydrobiopterin-dependent electron transfer in nitric oxide synthase regulates enzyme catalysis by additional mechanisms

Nitric oxide synthases (NOSs) are flavo-heme enzymes that require (6R)-tetrahydrobiopterin (H(4)B) for activity. Our single-catalytic turnover study with the inducible NOS oxygenase domain showed that a conserved Trp that interacts with H(4)B (Trp457 in mouse inducible NOS) regulates the kinetics of electron transfer between H(4)B and an enzyme heme-dioxy intermediate, and this in turn alters the kinetics and extent of Arg hydroxylation [Wang, Z.-Q., et al. (2001) Biochemistry 40, 12819-12825]. To investigate the impact of these effects on NADPH-driven NO synthesis by NOS, we generated and characterized the W457A mutant of inducible NOS and the corresponding W678A and W678F mutants of neuronal NOS. Mutant defects in protein solubility and dimerization were overcome by purifying them in the presence of sufficient Arg and H(4)B, enabling us to study their physical and catalytic profiles. Optical spectra of the ferric, ferrous, heme-dioxy, ferrous-NO, ferric-NO, and ferrous-CO forms of each mutant were similar to that of the wild type. However, the mutants had higher apparent K(m) values for H(4)B and in one mutant for Arg (W457A). They all had lower NO synthesis activities, uncoupled NADPH consumption, and a slower and less prominent buildup of enzyme heme-NO complex during steady-state catalysis. Further analyses showed the mutants had normal or near-normal heme midpoint potential and heme-NO complex reactivity with O(2), but had somewhat slower ferric heme reduction rates and markedly slower reactivities of their heme-dioxy intermediate. We conclude that the conserved Trp (1) has similar roles in two different NOS isozymes and (2) regulates delivery of both electrons required for O(2) activation (i.e., kinetics of ferric heme reduction by the NOS flavoprotein domain and reduction of the heme-dioxy intermediate by H(4)B). However, its regulation of H(4)B electron transfer is most important because this ensures efficient coupling of NADPH oxidation and NO synthesis by NOS.     

Direct evidence for nitric oxide production by a nitric-oxide synthase-like protein from Bacillus subtilis

Nitric-oxide synthases (NOSs) are widely distributed among prokaryotes and eukaryotes and have diverse functions in physiology. Recent genome sequencing revealed NOS-like protein in bacteria, but whether these proteins generate nitric oxide is unknown. We therefore cloned, expressed, and purified a NOS-like protein from Bacillus subtilis (bsNOS) and characterized its catalytic parameters in both multiple and single turnover reactions. bsNOS was dimeric, bound l-Arg and 6R-tetrahydrobiopterin with similar affinity as mammalian NOS, and generated nitrite from l-Arg when incubated with NADPH and a mammalian NOS reductase domain. Stopped-flow analysis showed that ferrous bsNOS reacted with O(2) to form a transient heme Fe(II)O(2) species in the presence of either Arg or the reaction intermediate N-hydroxy-l-arginine. In the latter case, disappearance of the Fe(II)O(2) species was kinetically and quantitatively coupled to formation of a transient heme Fe(III)NO product, which then dissociated to form ferric bsNOS. This behavior mirrors mammalian NOS enzymes and unambiguously shows that bsNOS can generate NO. NO formation required a bound tetrahydropteridine, and the kinetic effects of this cofactor were consistent with it donating an electron to the Fe(II)O(2) intermediate during the reaction. Dissociation of the heme Fe(III)NO product was much slower in bsNOS than in mammalian NOS. This constrains allowable rates of ferric heme reduction by a protein redox partner and underscores the utility of using a tetrahydropteridine electron donor in bsNOS.     

Regulation of inducible nitric oxide synthase by self-generated NO

A ferric heme-nitric oxide (NO) complex can build up in mouse inducible nitric oxide synthase (iNOS) during NO synthesis from L-arginine. We investigated its formation kinetics, effect on catalytic activity, dependence on solution NO concentration, and effect on enzyme oxygen response (apparent KmO2). Heme-NO complex formation was biphasic and was linked kinetically to an inhibition of electron flux and catalysis in iNOS. Experiments that utilized a superoxide generating system to scavenge NO showed that the magnitude of heme-NO complex formation directly depended on the NO concentration achieved in the reaction solution. However, a minor portion of heme-NO complex (20%) still formed during NO synthesis even when solution NO was completely scavenged. Formation of the intrinsic heme-NO complex, and the heme-NO complex related to buildup of solution NO, increased the apparent KmO2 of iNOS by 10- and 4-fold, respectively. Together, the data show heme-NO complex buildup in iNOS is due to both intrinsic NO binding and to equilibrium binding of solution NO, with the latter predominating when NO reaches high nanomolar to low micromolar concentrations. This behavior distinguishes iNOS from the other NOS isoforms and indicates a more complex regulation is possible for its activity and oxygen response in biologic settings.     

Thermodynamic and kinetic analysis of the nitrosyl, carbonyl, and dioxy heme complexes of neuronal nitric-oxide synthase. The roles of substrate and tetrahydrobiopterin in oxygen activation

Mammalian NO synthases catalyze the monooxygenation of L-arginine (L-Arg) to N-hydroxyarginine (NOHA) and the subsequent monooxygenation of this to NO and citrulline. Both steps proceed via formation of an oxyferrous heme complex and may ultimately lead to a ferrous NO complex, from which NO must be released. Electrochemical reduction of NO-bound neuronal nitricoxide synthase (nNOS) oxygenase domain was used to form the ferrous heme NO complex, which was found to be stable only in the presence of low NO concentrations, due to catalytic degradation of NO at the nNOS heme site. The reduction potential for the heme-NO complex was approximately -140 mV, which shifted to 0 mV in the presence of either L-Arg or NOHA. This indicates that the complex is stabilized by 14 kJ mol(-1) in the presence of substrate, consistent with a strong H-bonding interaction between NO and the guanidino group. Neither substrate influenced the reduction potential of the ferrous heme CO complex, however. Both L-Arg and NOHA appear to interact with bound NO in a similar way, indicating that both bind as guanidinium ions. The dissociation constant for NO bound to ferrous heme in the presence of l-Arg was determined electrochemically to be 0.17 nM, and the rate of dissociation was estimated to be 10(-4) s(-1), which is much slower than the rate of catalysis. Stopped-flow kinetic analysis of oxyferrous formation and decay showed that both l-Arg and NOHA also stabilize the ferrous heme dioxy complex, resulting in a 100-fold decrease in its rate of decay. Electron transfer from the active-site cofactor tetrahydrobiopterin (H4B) has been proposed to trigger the monoxygenation process. Consistent with this, substitution by the analogue/inhibitor 4-amino-H4B stabilized the oxyferrous complex by a further two orders of magnitude. H4B is required, therefore, to break down both the oxyferrousand ferrous nitrosyl complexes of nNOS during catalysis. The energetics of these processes necessitates an electron donor/acceptor operating within a specific reduction potential range, defining the role of H4B.     

Differences in three kinetic parameters underpin the unique catalytic profiles of nitric-oxide synthases I, II, and III.

We previously reported the existence of a special auto-regulation property of neuronal nitric-oxide synthase (NOS) based on NO near-geminate combination and partial trapping of neuronal NOS (nNOS) through a futile regenerating pathway. On this basis, we developed a kinetic simulation model that was proven to predict nNOS catalytic specificities and mutations effects (Santolini, J., Adak, S., Curran, C. M., and Stuehr, D. J. (2001) J. Biol. Chem. 276, 1233-1243; Adak, S., Santolini, J., Tikunova, S., Wang, Q., Johnson, J. D., and Stuehr, D. J. (2001) J. Biol. Chem. 276, 1244-1252). Here we show that the same model simulates and explains the distinct catalytic behaviors of inducible and endothelial NOS (iNOS and eNOS). Their marked differences were linked to variations in three basic parameters (rates of ferric heme reduction, ferric heme.NO dissociation, and ferrous heme.NO oxidation) that together control partitioning between futile and productive pathways and their relative rates. We also incorporated feedback inhibition into the kinetic model to account for potential rebinding of accumulated solution NO. The model accurately simulated the different relative impacts of both NOS.NO interactions (near-geminate combination of NO versus rebinding of solution NO) on catalytic behavior of each NOS isoform, including their speed and extent of heme.NO complex accumulation, K(m) for O(2), and propensity to transform NO into a higher oxide. Thus, individual catalytic behavior of any NOS can be understood through a single unified kinetic model. Because the model defines how different settings of individual kinetic parameters control regulation by two distinct NOS.NO interactions, it sheds light on mechanisms, structural features, and scope of NOS regulation and its physiologic impact.     

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.     

Comparative EPR studies on the nitrite reductases from Escherichia coli and Wolinella succinogenes

Hexaheme nitrite reductases purified to homogeneity from Escherichia coli K-12 and Wolinella succinogenes were studied by low-temperature EPR spectroscopy. In their isolated states, the two enzymes revealed nearly identical EPR spectra when measured at 12 K. Both high-spin and low-spin ferric heme EPR resonances with g values of 9.7, 3.7, 2.9, 2.3 and 1.5 were observed. These signals disappeared upon reduction by dithionite. Reaction of reduced enzyme with nitrite resulted in the formation of ferrous heme-NO complexes with distinct EPR spectral characteristics. The heme-NO complexes formed with the two enzymes differed, however, in g values and line-shapes. When reacted with hydroxylamine, reduced enzymes also showed the formation of ferrous heme-NO complexes. These results suggested the involvement of an enzyme-bound NO intermediate during the six-electron reduction of nitrite to ammonia catalyzed by these two hexaheme nitrite reductases. Heme proteins that can either expose bound NO to reduction or release it are significant components of both assimilatory and dissimilatory metabolisms of nitrate. The different ferrous heme-NO complexes detected for the two enzymes indicated, nevertheless, their subtle variation in heme reactivity during the reduction reaction.     

How does a valine residue that modulates heme-NO binding kinetics in inducible NO synthase regulate enzyme catalysis?

Nitric oxide (NO) release from nitric oxide synthases (NOSs) depends on the dissociation of a ferric heme-NO product complex (Fe^IIINO) that forms immediately after NO is made in the heme pocket. The NOS-like enzyme of Bacillus subtilis (bsNOS) has 10-20 fold slower Fe^IIINO dissociation rate (k_d) and NO association rate (k_on) compared to mammalian NOS counterparts. We previously showed that an Ile for Val substitution at the opening of the heme pocket in bsNOS contributes to these differences. The complementary mutation in mouse inducible NOS oxygenase domain (Val346Ile) decreased the NO k_on and k_d by 8 and 3-fold, respectively, compared to wild-type iNOSoxy, and also slowed the reductive processing of the heme-O₂ catalytic intermediate. To investigate how these changes affect steady-state catalytic behaviors, we generated and characterized the V346I mutant of full-length inducible NOS (iNOS). The mutant exhibited a 4-5 fold lower NO synthesis activity, an apparent uncoupled NADPH consumption, and formation of a heme-NO complex during catalysis that was no longer sensitive to solution NO scavenging. We found that these altered catalytic behaviors were not due to changes in the heme reduction rate or in the stability of the enzyme heme-O₂ intermediate, but instead were due to the slower NO k_on and k_d and a slower oxidation rate of the enzyme ferrous heme-NO complex. Computer simulations that utilized the measured kinetic values confirmed this interpretation, and revealed that the V346I iNOS has an enhanced NADPH-dependent NO dioxygenase activity that converts almost 1 NO to nitrate for every NO that the enzyme releases into solution. Together, our results highlight the importance of heme pocket geometry in tuning the NO release versus NO dioxygenase activities of iNOS.     

Molecular basis for hyperactivity in tryptophan 409 mutants of neuronal NO synthase

A ferrous heme-NO complex builds up in rat neuronal NO synthase during catalysis and lowers its activity. Mutation of a tryptophan located directly below the heme (Trp(409)) to Phe or Tyr causes hyperactive NO synthesis and less heme-NO complex buildup in the steady state (Adak, S., Crooks, C., Wang, Q., Crane, B. R., Tainer, J. A., Getzoff, E. D., and Stuehr, D. J. (1999) J. Biol. Chem. 274, 26907-26911). To understand the mechanism, we used conventional and stopped flow spectroscopy to compare kinetics of heme-NO complex formation, enzyme activity prior to and after complex formation, NO binding affinity, NO complex stability, and its reaction with O(2) in mutants and wild type nNOS. During the initial phase of NO synthesis, heme-NO complex formation was 3 and 5 times slower in W409F and W409Y, and their rates of NADPH oxidation were 50 and 30% that of wild type, probably due to slower heme reduction. NO complex formation slowed NADPH oxidation in the wild type by 7-fold but reduced mutant activities less than 2-fold, giving mutants higher final activities. NO binding kinetics were similar among mutants and wild type, although in ferrous W409Y (and to a lesser extent W409F) the 436-nm NO complex converted to a 417-nm NO complex with time. Oxidation of the ferrous heme-NO complex to ferric enzyme was 7 times faster in Trp(409) mutants than in wild type. Thus, mutant hyperactivity derives from slower formation and faster decay of the heme-NO complex. Together, these minimize partitioning into the NO-bound form.     

Electron paramagnetic resonance characterization of tetrahydrobiopterin radical formation in bacterial nitric oxide synthase compared to mammalian nitric oxide synthase

H(4)B is an essential catalytic cofactor of the mNOSs. It acts as an electron donor and activates the ferrous heme-oxygen complex intermediate during Arg oxidation (first step) and NOHA oxidation (second step) leading to nitric oxide and citrulline as final products. However, its role as a proton donor is still debated. Furthermore, its exact involvement has never been explored for other NOSs such as NOS-like proteins from bacteria. This article proposes a comparative study of the role of H(4)B between iNOS and bsNOS. In this work, we have used freeze-quench to stop the arginine and NOHA oxidation reactions and trap reaction intermediates. We have characterized these intermediates using multifrequency electron paramagnetic resonance. For the first time, to our knowledge, we report a radical formation for a nonmammalian NOS. The results indicate that bsNOS, like iNOS, has the capacity to generate a pterin radical during Arg oxidation. Our current electron paramagnetic resonance data suggest that this radical is protonated indicating that H(4)B may not transfer any proton. In the 2nd step, the radical trapped for iNOS is also suggested to be protonated as in the 1st step, whereas it was not possible to trap a radical for the bsNOS 2nd step. Our data highlight potential differences for the catalytic mechanism of NOHA oxidation between mammalian and bacterial NOSs.     

Chimeras of nitric-oxide synthase types I and III establish fundamental correlates between heme reduction, heme-NO complex formation, and catalytic activity

Neuronal nitric-oxide synthase (nNOS or NOS I) and endothelial NOS (eNOS or NOS III) differ widely in their reductase and nitric oxide (NO) synthesis activities, electron transfer rates, and propensities to form a heme-NO complex during catalysis. We generated chimeras by swapping eNOS and nNOS oxygenase domains to understand the basis for these differences and to identify structural elements that determine their catalytic behaviors. Swapping oxygenase domains did not alter domain-specific catalytic functions (cytochrome c reduction or H(2)O(2)-supported N(omega)-hydroxy-l-arginine oxidation) but markedly affected steady-state NO synthesis and NADPH oxidation compared with native eNOS and nNOS. Stopped-flow analysis showed that reductase domains either maintained (nNOS) or slightly exceeded (eNOS) their native rates of heme reduction in each chimera. Heme reduction rates were found to correlate with the initial rates of NADPH oxidation and heme-NO complex formation, with the percentage of heme-NO complex attained during the steady state, and with NO synthesis activity. Oxygenase domain identity influenced these parameters to a lesser degree. We conclude: 1) Heme reduction rates in nNOS and eNOS are controlled primarily by their reductase domains and are almost independent of oxygenase domain identity. 2) Heme reduction rate is the dominant parameter controlling the kinetics and extent of heme-NO complex formation in both eNOS and nNOS, and thus it determines to what degree heme-NO complex formation influences their steady-state NO synthesis, whereas oxygenase domains provide minor but important influences. 3) General principles that relate heme reduction rate, heme-NO complex formation, and NO synthesis are not specific for nNOS but apply to eNOS as well.     

Importance of valine 567 in substrate recognition and oxidation by neuronal nitric oxide synthase

Nitric oxide (NO) is synthesised by a two-step oxidation of -arginine (L-Arg) in the active site of nitric oxide synthase (NOS) with formation of an intermediate, N omega-hydroxy-L-Arg (NOHA). Crystal structures of NOSs have shown the importance of an active-site Val567 residue (numbered for rat neuronal NOS, nNOS) interacting with non-amino acid substrates. To investigate the role of this Val residue in substrate recognition and NO-formation activity by nNOS, we generated and purified four Val567 mutants of nNOS, Val567Leu, Val567Phe, Val567Arg and Val567Glu. We characterized these proteins and tested their ability to generate NO from the oxidation of natural substrates L-Arg and NOHA, and from N-hydroxyguanidines previously identified as alternative substrates for nNOS. The Val567Leu mutant displayed lower NO formation activities than the wild type (WT) in the presence of all tested compounds. Surprisingly, the Val567Phe mutant formed low amounts of NO only from NOHA. These two mutants displayed lower affinity for L-Arg and NOHA than the WT protein. Val576Glu and Val567Arg mutants were much less stable and did not lead to any formation of NO. These results suggest that Val567 is an important residue for preserving the integrity of the active site, for substrate binding, and subsequently for NO-formation in nNOS.