A proximal tryptophan in NO synthase controls activity by a novel mechanism

The heme of neuronal nitric oxide synthase (nNOS) participates in O2 activation but also binds self-generated NO, resulting in reversible feedback inhibition. We utilized mutagenesis to investigate if a conserved tryptophan residue (Trp409), which engages in pi-stacking with the heme and hydrogen bonds to its axial cysteine ligand, helps control catalysis and regulation by NO. Mutants W409F and W409Y were hyperactive regarding NO synthesis without affecting cytochrome c reduction, reductase-independent N-hydroxyarginine oxidation, or Arg and tetrahydrobiopterin binding. In the absence of Arg electron flux through the heme was slower in the W409 mutants than in wild-type. However, less NO complex accumulated during NO synthesis by the mutants. To understand the mechanism, we compared the kinetics of heme-NO complex formation, rate of heme reduction, kcat prior to and after NO complex formation, NO binding affinity, NO complex stability, and its reaction with O2. During the initial phase of NO synthesis, heme-NO complex formation was three and five times slower in W409F and W409Y, which corresponded to a slower heme reduction. NO complex formation inhibited wild-type turnover 7-fold but reduced mutant turnover less than 2-fold, giving mutants higher steady-state activities. NO binding kinetics were similar among mutants and wild type, although mutants also formed a 417 nm ferrous-NO complex. Oxidation of ferrous-NO complex was seven times faster in mutants than in wild type. We conclude that mutant hyperactivity primarily derives from slower heme reduction and faster oxidation of the heme-NO complex by O2. In this way Trp409 mutations minimize NO feedback inhibition by limiting buildup of the ferrous-NO complex during the steady state. Conservation of W409 among NOS suggests that this proximal Trp may regulate NO feedback inhibition and is important for enzyme physiologic function.     

Aromatic residues and neighboring Arg414 in the (6R)-5,6,7, 8-tetrahydro-L-biopterin binding site of full-length neuronal nitric-oxide synthase are crucial in catalysis and heme reduction with NADPH

Nitric-oxide synthase (NOS) requires the cofactor, (6R)-5,6,7, 8-tetrahydrobiopterin (H4B), for catalytic activity. The crystal structures of NOSs indicate that H4B is surrounded by aromatic residues. We have mutated the conserved aromatic acids, Trp(676), Trp(678), Phe(691), His(692), and Tyr(706), together with the neighboring Arg(414) residue within the H4B binding region of full-length neuronal NOS. The W676L, W678L, and F691L mutants had no NO formation activity and had very low heme reduction rates (<0.02 min(-1)) with NADPH. Thus, it appears that Trp(676), Trp(678), and Phe(691) are important to retain the appropriate active site conformation for H4B/l-Arg binding and/or electron transfer to the heme from NADPH. The mutation of Tyr(706) to Leu and Phe decreased the activity down to 13 and 29%, respectively, of that of the wild type together with a dramatically increased EC(50) value for H4B (30-40-fold of wild type). The Tyr(706) phenol group interacts with the heme propionate and Arg(414) amine via hydrogen bonds. The mutation of Arg(414) to Leu and Glu resulted in the total loss of NO formation activity and of the heme reduction with NADPH. Thus, hydrogen bond networks consisting of the heme carboxylate, Tyr(706), and Arg(414) are crucial in stabilizing the appropriate conformation(s) of the heme active site for H4B/l-Arg binding and/or efficient electron transfer to occur.     

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.     

Crucial role of Lys(423) in the electron transfer of neuronal nitric-oxide synthase.

Nitric-oxide synthase (NOS) is composed of an oxygenase domain having cytochrome P450-type heme active site and a reductase domain having FAD- and FMN-binding sites. To investigate the route of electron transfer from the reductase domain to the heme, we generated mutants at Lys(423) in the heme proximal site of neuronal NOS and examined the catalytic activities, electron transfer rates, and NADPH oxidation rates. A K423E mutant showed no NO formation activity (<0.1 nmol/min/nmol heme), in contrast with that (72 nmol/min/nmol heme) of the wild type enzyme. The electron transfer rate (0.01 min(-1)) of the K423E on addition of excess NADPH was much slower than that (>10 min(-1)) of the wild type enzyme. From the crystal structure of the oxygenase domain of endothelial NOS, Lys(423) of neuronal NOS is likely to interact with Trp(409) which lies in contact with the heme plane and with Cys(415), the axial ligand. It is also exposed to solvent and lies in the region where the heme is closest to the protein surface. Thus, it seems likely that ionic interactions between Lys(423) and the reductase domain may help to form a flavin to heme electron transfer pathway.     

Critical role of the neuronal nitric-oxide synthase heme proximal side residue, Arg418, in catalysis and electron transfer.

Nitric oxide (NO) is synthesized from L-Arg in the P450-type heme active site of nitric-oxide synthase (NOS). The internal axial ligand of the heme, Cys415, may hydrogen-bond to the side chain of the conserved Arg418 residue in neuronal NOS (nNOS). To understand the role of Arg418, we generated the nNOS mutants, Arg418Ala and Arg418Leu. NO formation activities with the mutants using both L-Arg and NHA as substrates were less than 0.1 nmol/min/nmol heme, in contrast to rates of 34-35 nmol/min/nmol heme with the wild-type enzyme. The heme reduction rate of the mutants was very slow, less than 10(-2) min(-1), in contrast with that (more than 10 min(-1)) of the wild type. The backbone amide group of Arg418 interacts with the Cys415 thiolate through van der Waals contact, whereas the carbonyl oxygen of Cys415 and the guanidino N(epsilon) atom of Arg418 form a tight hydrogen bond. The results suggest that Arg418 is critical in preserving the heme proximal structure and thus, is indirectly involved in both catalysis and electron transfer from the reductase domain to the heme.     

Regulation of the properties of the heme-NO complexes in nitric-oxide synthase by hydrogen bonding to the proximal cysteine

Nitric-oxide synthase (NOS) catalyzes the formation of NO and citrulline from l-arginine and oxygen. However, the NO so formed has been found to auto-inhibit the enzymatic activity significantly. We hypothesized that the NO reactivity is in part controlled by hydrogen bonding between the conserved tryptophan residue (position 409 in the neuronal isoform of NOS (nNOS)) and the cysteine residue that forms the proximal bond to the heme. By using resonance Raman spectroscopy and NO as a probe of the heme environment, we show that in the W409F and W409Y mutants of the oxygenase domain of the neuronal enzyme (nNOSox), the Fe-NO bond in the Fe3+NO complex is weaker than in the wild type enzyme, consistent with the loss of a hydrogen bond on the sulfur atom of the proximal cysteine residue. The weaker Fe-NO bond in the W409F and W409Y mutants might result in a faster rate of NO dissociation from the ferric heme in the Trp-409 mutants as compared with the wild type enzyme, which could contribute to the lower accumulation of the inhibitory NO-bound complexes observed during catalysis with the Trp-409 mutants (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). The optical and resonance Raman spectra of the Fe2+NO complexes of the Trp-409 mutants differ from those of the wild type enzyme and indicate that a significant population of a five-coordinate Fe2+NO complex is present. These data show that the hydrogen bond provided by the Trp-409 residue is necessary to maintain the thiolate coordination when NO binds to the ferrous heme. Taken together our results indicate that the heme environment on the proximal side of nNOS is critical for the formation of a stable iron-cysteine bond and for the control of the electronic properties of heme-NO complexes.     

The 42-amino acid insert in the FMN domain of neuronal nitric-oxide synthase exerts control over Ca(2+)/calmodulin-dependent electron transfer.

The neuronal and endothelial nitric-oxide synthases (nNOS and eNOS) differ from inducible NOS in their dependence on the intracellular Ca(2+) concentration. Both nNOS and eNOS are activated by the reversible binding of calmodulin (CaM) in the presence of Ca(2+), whereas inducible NOS binds CaM irreversibly. One major divergence in the close sequence similarity between the NOS isoforms is a 40-50-amino acid insert in the middle of the FMN-binding domains of nNOS and eNOS. It has previously been proposed that this insert forms an autoinhibitory domain designed to destabilize CaM binding and increase its Ca(2+) dependence. To examine the importance of the insert we constructed two deletion mutants designed to remove the bulk of it from nNOS. Both mutants (Delta40 and Delta42) retained maximal NO synthesis activity at lower concentrations of free Ca(2+) than the wild type enzyme. They were also found to retain 30% of their activity in the absence of Ca(2+)/CaM, indicating that the insert plays an important role in disabling the enzyme when the physiological Ca(2+) concentration is low. Reduction of nNOS heme by NADPH under rigorous anaerobic conditions was found to occur in the wild type enzyme only in the presence of Ca(2+)/CaM. However, reduction of heme in the Delta40 mutant occurred spontaneously on addition of NADPH in the absence of Ca(2+)/CaM. This suggests that the insert regulates activity by inhibiting electron transfer from FMN to heme in the absence of Ca(2+)/CaM and by destabilizing CaM binding at low Ca(2+) concentrations, consistent with its role as an autoinhibitory domain.     

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.     

Unusual role of Tyr588 of neuronal nitric oxide synthase in controlling substrate specificity and electron transfer

Nitric oxide (NO) is synthesized from l-Arg via N(G)-hydroxyl-l-Arg (NHA) in the heme active site of nitric oxide synthase (NOS). According to the crystal structure of other NOS isoforms, the carboxylate group of l-Arg hydrogen bonds to the hydroxyl group of the conserved Tyr588 residue in the heme distal site of neuronal NOS (nNOS). Indeed, the nNOS mutations Tyr588His, Tyr588Ser, and Tyr588Phe markedly increased the dissociation constants for l-Arg and NHA by 2.2-8.2-fold and 1.5-3.9-fold, respectively. Similarly, Tyr588His and Tyr588Ser mutations markedly decreased the l-Arg-driven NO formation rates by 50 and 30% than that of the wild type, respectively. However, the catalytic activities of the same mutants using NHA were higher than that of the wild type by up to 136%. As a result, the turnover ratio of NHA to l-Arg was 4.12 for the Tyr588Ser mutant, compared with 1.07 for the wild-type enzyme. Intriguingly, heme reduction rates for the Tyr588 mutants were much lower than for wild type by two orders of magnitude.     

Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia.

Nitric oxide (NO) plays an important role in the pathogenesis of neuronal injury during cerebral ischemia. The endothelial and neuronal isoforms of nitric oxide synthase (eNOS, nNOS) generate NO, but NO generation from these two isoforms can have opposing roles in the process of ischemic injury. While increased NO production from nNOS in neurons can cause neuronal injury, endothelial NO production from eNOS can decrease ischemic injury by inducing vasodilation. However, the relative magnitude and time course of NO generation from each isoform during cerebral ischemia has not been previously determined. Therefore, electron paramagnetic resonance spectroscopy was applied to directly detect NO in the brain of mice in the basal state and following global cerebral ischemia induced by cardiac arrest. The relative amount of NO derived from eNOS and nNOS was accessed using transgenic eNOS(-/-) or nNOS(-/-) mice and matched wild-type control mice. NO was trapped using Fe(II)-diethyldithiocarbamate. In wild-type mice, only small NO signals were seen prior to ischemia, but after 10 to 20 min of ischemia the signals increased more than 4-fold. This NO generation was inhibited more than 70% by NOS inhibition. In either nNOS(-/-) or eNOS(-/-) mice before ischemia, NO generation was decreased about 50% compared to that in wild-type mice. Following the onset of ischemia a rapid increase in NO occurred in nNOS(-/-) mice peaking after only 10 min. The production of NO in the eNOS(-/-) mice paralleled that in the wild type with a progressive increase over 20 min, suggesting progressive accumulation of NO from nNOS following the onset of ischemia. NOS activity measurements demonstrated that eNOS(-/-) and nNOS(-/-) brains had 90% and < 10%, respectively, of the activity measured in wild type. Thus, while eNOS contributes only a fraction of total brain NOS activity, during the early minutes of cerebral ischemia prominent NO generation from this isoform occurs, confirming its importance in modulating the process of ischemic injury.     

Engineering out motion: a surface disulfide bond alters the mobility of tryptophan 22 in cytochrome b5 as probed by time-resolved fluorescence and 1H NMR experiments

In the accompanying paper [Storch et al. (1999) Biochemistry 38, 5054-5064] equilibrium denaturation studies and molecular dynamics (MD) simulations were used to investigate localized dynamics on the surface of cytochrome b5 (cyt b5) that result in the formation of a cleft. In those studies, an S18C:R47C disulfide mutant was engineered to inhibit cleft mobility. Temperature- and urea-induced denaturation studies revealed significant differences in Trp 22 fluorescence between the wild-type and mutant proteins. On the basis of the results, it was proposed that wild type populates a conformational ensemble that is unavailable to the disulfide mutant and is mediated by cleft mobility. As a result, the solvent accessibility of Trp 22 is decreased in S18C:R47C, suggesting that the local environment of this residue is less mobile due to the constraining effects of the disulfide on cleft dynamics. To further probe the structural effects on the local environment of Trp 22 caused by inhibition of cleft formation, we report here the results of steady-state and time-resolved fluorescence quenching, differential phase/modulation fluorescence anisotropy, and 1H NMR studies. In Trp fluorescence experiments, the Stern-Volmer quenching constant increases in wild type versus the oxidized disulfide mutant with increasing temperature. At 50 degrees C, KSV is nearly 1.5-fold greater in wild type compared to the oxidized disulfide mutant. In the reduced disulfide mutant, KSV was the same as wild type. The bimolecular collisional quenching constant, kq, for acrylamide quenching of Trp 22 increases 2.7-fold for wild type and only 1.8-fold for S18C:R47C, upon increasing the temperature from 25 to 50 degrees C. The time-resolved anisotropy decay at 25 degrees C was fit to a double-exponential decay for both the wild type and S18C:R47C. Both proteins exhibited a minor contribution from a low-amplitude fast decay, consistent with local motion of Trp 22. This component was more prevalent in the wild type, and the fractional contribution increased significantly upon raising the temperature. The fast rotational component of the S18C:R47C mutant was less sensitive to increasing temperature. A comparison of the 1H NMR monitored temperature titration of the delta-methyl protons of Ile 76 for wild type and oxidized disulfide mutant, S18C:R47C, showed a significantly smaller downfield shift for the mutant protein, suggesting that Trp 22 in the mutant protein experiences comparatively decreased cleft dynamics in core 2 at higher temperatures. Furthermore, comparison of the delta'-methyl protons of Leu 25 in the two proteins revealed a difference in the ratio of the equilibrium heme conformers of 1.2:1 for S18C:R47C versus 1.5:1 for wild type at 40 degrees C. The difference in equilibrium heme orientations between wild type and S18C:R47C suggests that the disulfide bond affects heme binding within core 1, possibly through damped cleft fluctuations. Taken together, the NMR and fluorescence studies support the proposal that an engineered disulfide bond inhibits the formation of a dynamic cleft on the surface of cyt b5.     

Tryptophan 409 controls the activity of neuronal nitric-oxide synthase by regulating nitric oxide feedback inhibition.

The heme of neuronal nitric-oxide synthase participates in oxygen activation but also binds self-generated NO during catalysis resulting in reversible feedback inhibition. We utilized point mutagenesis to investigate if a conserved tryptophan residue (Trp-409), which engages in pi-stacking with the heme and hydrogen bonds to its axial cysteine ligand, helps control catalysis and regulation by NO. Surprisingly, mutants W409F and W409Y were hyperactive compared with the wild type regarding NO synthesis without affecting cytochrome c reduction, reductase-independent N-hydroxyarginine oxidation, or Arg and tetrahydrobiopterin binding. In the absence of Arg, NADPH oxidation measurements showed that electron flux through the heme was actually slower in the Trp-409 mutants than in wild-type nNOS. However, little or no NO complex accumulated during NO synthesis by the mutants, as opposed to the wild type. This difference was potentially related to mutants forming unstable 6-coordinate ferrous-NO complexes under anaerobic conditions even in the presence of Arg and tetrahydrobiopterin. Thus, Trp-409 mutations minimize NO feedback inhibition by preventing buildup of an inactive ferrous-NO complex during the steady state. This overcomes the negative effect of the mutation on electron flux and results in hyperactivity. Conservation of Trp-409 among different NOS suggests that the ability of this residue to regulate heme reduction and NO complex formation is important for enzyme physiologic function.     

Control of Electron Transfer and Catalysis in Neuronal Nitric-oxide Synthase (nNOS) by a Hinge Connecting Its FMN and FAD-NADPH Domains

In nitric-oxide synthases (NOSs), two flexible hinges connect the FMN domain to the rest of the enzyme and may guide its interactions with partner domains for electron transfer and catalysis. We investigated the role of the FMN-FAD/NADPH hinge in rat neuronal NOS (nNOS) by constructing mutants that either shortened or lengthened this hinge by 2, 4, and 6 residues. Shortening the hinge progressively inhibited electron flux through the calmodulin (CaM)-free and CaM-bound nNOS to cytochrome c, whereas hinge lengthening relieved repression of electron flux in CaM-free nNOS and had no impact or slowed electron flux through CaM-bound nNOS to cytochrome c. How hinge length influenced heme reduction depended on whether enzyme flavins were pre-reduced with NADPH prior to triggering heme reduction. Without pre-reduction, changing the hinge length was deleterious; with pre-reduction, the hinge shortening was deleterious, and hinge lengthening increased heme reduction rates beyond wild type. Flavin fluorescence and stopped-flow kinetic studies on CaM-bound enzymes suggested hinge lengthening slowed the domain-domain interaction needed for FMN reduction. All hinge length changes lowered NO synthesis activity and increased uncoupled NADPH consumption. We conclude that several aspects of catalysis are sensitive to FMN-FAD/NADPH hinge length and that the native hinge allows a best compromise among the FMN domain interactions and associated electron transfer events to maximize NO synthesis and minimize uncoupled NADPH consumption.     

Conversion of the proximal histidine ligand to glutamine restores activity to an inactive mutant of cytochrome c peroxidase.

Using site-directed mutagenesis, a double mutant in yeast cytochrome c peroxidase (CCP) has been constructed where the proximal ligand, His175, has been converted to glutamine and the neighboring Trp191 has been converted to phenylalanine. The refined 2.4-A crystal structure of the double mutant shows that the Gln175 side chain is within coordination distance of the heme iron atom and that Phe191 occupies the same position as Trp191 in the native enzyme with very little rearrangement outside the immediate vicinity of the mutations. Consistent with earlier work, we find that the single mutant, His175-->Gln, is fully active under steady state assay conditions and that as reported earlier (Mauro et al., 1988), the Trp191-->Phe mutant exhibits only < 0.05% activity. However, the double mutant, His175-->Gln/Phe191-->Phe, exhibits 20% wild type activity. Since it is known that the Trp191-->Phe mutant is inactive because it can no longer transfer electrons from ferrocytochrome c, changing the nature of the proximal ligand is able to restore this activity. These results raise interesting questions regarding the mechanism of interprotein electron transfer reactions.     

Kinetics of CO and NO ligation with the Cys(331)-->Ala mutant of neuronal nitric-oxide synthase

Nitric-oxide synthases (NOS) catalyze the conversion of l-arginine to NO, which then stimulates many physiological processes. In the active form, each NOS is a dimer; each strand has both a heme-binding oxygenase domain and a reductase domain. In neuronal NOS (nNOS), there is a conserved cysteine motif (CX(4)C) that participates in a ZnS(4) center, which stabilizes the dimer interface and/or the flavoprotein-heme domain interface. Previously, the Cys(331) --> Ala mutant was produced, and it proved to be inactive in catalysis and to have structural defects that disrupt the binding of l-Arg and tetrahydrobiopterin (BH(4)). Because binding l-Arg and BH(4) to wild type nNOS profoundly affects CO binding with little effect on NO binding, ligand binding to the mutant was characterized as follows. 1) The mutant initially has behavior different from native protein but reminiscent of isolated heme domain subchains. 2) Adding l-Arg and BH(4) has little effect immediately but substantial effect after extended incubation. 3) Incubation for 12 h restores behavior similar but not quite identical to that of wild type nNOS. Such incubation was shown previously to restore most but not all catalytic activity. These kinetic studies substantiate the hypothesis that zinc content is related to a structural rather than a catalytic role in maintaining active nNOS.     

Distal residue-CO interaction in carbonmonoxy myoglobins: a molecular dynamics study of three distal mutants.

Six 90-ps molecular dynamics trajectories, two for each of three distal mutants of sperm whale carbonmonoxy myoglobin, are reported; solvent waters within 16 A of the active site have been included. In both His64GIn trajectories, the distal side chain remains part of the heme pocket, forming a "closed" conformation similar to that of the wild type 64N delta H tautomer. Despite a connectivity more closely resembling the N epsilon H histidine tautomer, close interactions with the carbonyl ligand similar to those observed for the wild type 64N epsilon H tautomer are prevented in this mutant by repulsive interactions between the carbonyl O and the 64O epsilon. The aliphatic distal side chain of the His64Leu mutant shows little interaction with the carbonyl ligand in either His64Leu trajectory. Solvent water molecules move into and out of the active site in the His64Gly mutant trajectories; during all the other carbonmonoxy myoglobin trajectories, including the wild type distal tautomers considered in an earlier work, solvent molecules rarely encroach closer than 6 A of the active site. These results are consistent with a recent structural interpretation of the wild type infrared spectrum, and the current reinterpretation that the distal-ligand interaction in carbonmonoxy myoglobin is largely electrostatic, not steric, in nature.     

Zinc content of Escherichia coli-expressed constitutive isoforms of nitric-oxide synthase. Enzymatic activity and effect of pterin.

Recently, we obtained x-ray crystallographic data showing the presence of a ZnS4 center in the structure of Escherichia coli-expressed bovine endothelial nitric-oxide synthase (eNOS) and rat neuronal nitric-oxide synthase (nNOS). The zinc atom is coordinated by two CXXXXC motifs, one motif being contributed by each NOS monomer (cysteine 326 through cysteine 331 in rat nNOS). Mutation of the nNOS cysteine 331 to alanine (C331A) results in the loss of NO. synthetic activity and also results in an inability to bind zinc efficiently. Although prolonged incubation of the C331A mutant of nNOS with high concentrations of L-arginine results in a catalytically active enzyme, zinc binding is not restored. In this study, we investigate the zinc stoichiometry in wild-type nNOS and eNOS, as well as in the C331A-mutated nNOS, using a chelation assay and electrothermal vaporization-inductively coupled plasma-mass spectrometry. The data reveal an approximate 2:1 stoichiometry of heme to zinc in (6R)-5,6,7,8-tetrahydro-L-biopterin-replete, wild-type nNOS and eNOS and show that the reactivated C331A mutant of nNOS has a limited ability to bind zinc. The present study substantiates that the zinc in NOS is structural rather than catalytic and is important for maintaining optimally functional, enzymatically active, constitutive NOSs.     

A structural role for tryptophan 188 of inducible nitric oxide synthase

All nitric oxide synthase (NOS) isotypes bear a conserved tryptophan that stacks against the proximal face of the heme cofactor. Recently two hyperactive variants of neuronal NOS were reported in which this residue (W409) was replaced by phenylalanine or tyrosine. We find that mutation of the same residue in the oxygenase domain of inducible NOS (W188) to phenylalanine causes severe destabilization of heme binding. W188F is isolated in a predominantly heme-free state, and axial thiolate ligation to the residual bound heme is unstable. However, W188F is soluble and is expressed at levels comparable to wild type. While circular dichroism spectroscopy demonstrates the loss of some secondary structure, the protein chain is not completely denatured and it retains much of its fold between pH 7.5 and 4. This proximal tryptophan of NOS represents a case where a residue is conserved within an enzyme family but for distinct purposes that are isotype-dependent.     

Role of tryptophan 121 in the soluble CuA domain of cytochrome c oxidase: structure and electron transfer studies

To investigate the contribution of tryptophan-121 (Trp121) residue to the structure and function of soluble CuA domain of cytochrome c oxidase, three mutant proteins, Trp121Tyr, Trp121Leu and Trp121-deleted mutant of the soluble domain of Paracoccus versutus cytochrome c oxidase, were constructed and expressed in Escherichia coli BL21 (DE3). Optical spectral studies showed that both the coordination structure of the CuA center and the secondary structure of the protein were changed significantly in the Leu substitution and deletion mutants of Trp121. Their electron transfer activity with cytochrome c was inhibited severely, as shown in stopped-flow kinetic studies. However, the CuA center can be reconstructed in the Trp121Tyr mutant although its stability decreases compared with the wild-type protein. This mutant keeps the same secondary structure as the wild-type protein, but can only transfer electrons with cytochrome c at a rate of one-seventh-fold. Based on the information on the structure, we also investigated and analyzed the possible factors that affect electron transfer. It appears that the aromatic ring, the size of the side chain and the hydrogen bonding ability of the Trp121 are crucial to the structure and function of the soluble CuA domain.     

Renal cortical and medullary blood flow responses to L-NAME and ANG II in wild-type, nNOS null mutant, and eNOS null mutant mice

Experiments in wild-type (WT; C57BL/6J) mice, endothelial nitric oxide synthase null mutant [eNOS(-/-)] mice, and neuronal NOS null mutant [nNOS(-/-)] mice were performed to determine which NOS isoform regulates renal cortical and medullary blood flow under basal conditions and during the infusion of ANG II. Inhibition of NOS with N(omega)-nitro-l-arginine methyl ester (l-NAME; 50 mg/kg iv) in Inactin-anesthetized WT and nNOS(-/-) mice increased arterial blood pressure by 28-31 mmHg and significantly decreased blood flow in the renal cortex (18-24%) and the renal medulla (13-18%). In contrast, blood pressure and renal cortical and medullary blood flow were unaltered after l-NAME administration to eNOS(-/-) mice, indicating that NO derived from eNOS regulates baseline vascular resistance in mice. In subsequent experiments, intravenous ANG II (20 ng x kg(-1) x min(-1)) significantly decreased renal cortical blood flow (by 15-25%) in WT, eNOS(-/-), nNOS(-/-), and WT mice treated with l-NAME. The infusion of ANG II, however, led to a significant increase in medullary blood flow (12-15%) in WT and eNOS(-/-) mice. The increase in medullary blood flow following ANG II infusion was not observed in nNOS(-/-) mice, in WT or eNOS(-/-) mice pretreated with l-NAME, or in WT mice administered the nNOS inhibitor 5-(1-imino-3-butenyl)-l-ornithine (1 mg x kg(-1) x h(-1)). These data demonstrate that NO from eNOS regulates baseline blood flow in the mouse renal cortex and medulla, while NO produced by nNOS mediates an increase in medullary blood flow in response to ANG II.