Molecular Basis for Hyperactivity in Tryptophan 409 Mutants of Neuronal NO Synthase (original) (raw)
Related papers
A proximal tryptophan in NO synthase controls activity by a novel mechanism
Journal of Inorganic Biochemistry, 2001
The heme of neuronal nitric oxide synthase (nNOS) participates in O activation but also binds self-generated NO, resulting in 2 reversible feedback inhibition. We utilized mutagenesis to investigate if a conserved tryptophan residue (Trp409), which engages in p-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, k prior to and after NO complex formation, NO cat binding affinity, NO complex stability, and its reaction with O . During the initial phase of NO synthesis, heme-NO complex formation 2 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 O . In this way Trp409 mutations minimize NO feedback inhibition by 2 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.
Journal of Biological Chemistry, 1999
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 -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.
A Structural Role for Tryptophan 188 of Inducible Nitric Oxide Synthase
Biochemical and Biophysical Research Communications, 2001
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.
Biochemistry, 2005
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. domain of inducible nitric oxide synthase; nNOSoxy, oxygenase domain of neuronal nitric oxide synthase; Arg, L-arginine; DTT, dithiothreitol; NO, nitric oxide; EPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid; NOHA, N ω -hydroxy-L-arginine; H4B, (6R)-5,6,7,8-tetrahydro-Lbiopterin; Im, imidazole; CaM, calmodulin; FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide phosphate; Fe II , ferrous species; Fe III , ferric species; Fe II O2, ferrous heme-dioxy species. In referring to amino acids, we use the three-letter abbreviation, for example, Trp678. In referring to amino acid mutations, we use one-letter symbols to indicate the specific mutation, for example, W678A.
Journal of Biological Chemistry, 2001
We mutated a target residue in that site (Ser-1412 to Asp) to mimic phosphorylation and then characterized the mutant using conventional and stopped-flow spectroscopies. Compared with wild-type, S1412D nNOS catalyzed faster cytochrome c and ferricyanide reduction but displayed slower steady-state NO synthesis with greater uncoupling of NADPH oxidation. Paradoxically, the mutant had faster heme reduction, faster heme-NO complex formation, and greater heme-NO complex accumulation at steady state. To understand how these behaviors related to flavin and heme reduction rates, we utilized three soybean calmodulins (CaMs) that supported a range of slower flavin and heme reduction rates in mutant and wild-type nNOS. Reductase activity and two catalytic parameters (speed and amount of heme-NO complex formation) related directly to the speed of flavin and heme reduction. In contrast, steadystate NO synthesis increased, reached a plateau, and then fell at the highest rate of heme reduction that was obtained with S1412D nNOS ؉ CaM. Substituting with soybean CaM slowed heme reduction and increased steady-state NO synthesis by the mutant. We conclude the following. 1) The S1412D mutation speeds electron transfer out of the reductase domain. 2) Faster heme reduction speeds intrinsic NO synthesis but diminishes NO release in the steady state. 3) Heme reduction displays an optimum regarding NO release during steady state. The unique behavior of S1412D nNOS reveals the importance of heme reduction rate in controlling steady-state activity and suggests that nNOS already has a near-optimal rate of heme reduction.
Control of electron transfer in neuronal NO synthase
Biochemical Society Transactions, 2001
The nitric oxide synthases (NOSs) are dimeric flavocytochromes consisting of an oxygenase domain with cytochrome P450-like Cys-ligated haem, coupled to a diflavin reductase domain, which is related to cytochrome P450 reductase. The NOSs catalyse the sequential mono-oxygenation of arginine to N-hydroxyarginine and then to citrulline and NO. The constitutive NOS isoforms (cNOSs) are regulated by calmodulin (CaM), which binds at elevated concentrations of free Ca2+, whereas the inducible isoform binds CaM irreversibly. One of the main structural differences between the constitutive and inducible isoforms is an insert of 40–50 amino acids in the FMN-binding domain of the cNOSs. Deletion of the insert in rat neuronal NOS (nNOS) led to a mutant enzyme which binds CaM at lower Ca2+ concentrations and which retains activity in the absence of CaM. In order to resolve the mechanism of action of CaM activation we determined reduction potentials for the FMN and FAD cofactors of rat nNOS in the ...
Journal of the American Chemical Society, 1998
The nitric oxide synthases (NOS) are heme-containing enzymes responsible for catalyzing the fiveelectron oxidation of a guanidino nitrogen of L-arginine to produce the free radical nitric oxide. The binding sites of the heme group, as well as of the L-arginine substrate and tetrahydrobiopterin cofactor, are located within the oxygenase domain of the NOS enzymes. Reduction of the heme is the first committed step in catalysis, as this allows for binding and activation of molecular oxygen, followed by oxidative attack on the L-arginine substrate. As with heme groups in other enzymes, the electronic properties of the NOS heme are modified by substrate and cofactor binding in its vicinity. Here we present the first quantitative thermodynamic data of the NOS heme with the determination of the heme midpoint reduction potentials for the neuronal NOS and inducible NOS oxygenase domains. In the absence of L-arginine and tetrahydrobiopterin, the midpoint potential of the inducible NOS oxygenase heme iron is over 100 mV lower than that of the neuronal NOS oxygenase heme iron. Binding of the substrate alone, cofactor alone, or both combined with the inducible NOS oxygenase increases the heme iron reduction potential by 112, 52, and 84 mV, respectively. On the basis of these data, we calculate that the binding affinities of L-arginine and tetrahydrobiopterin increase by about 80-fold and 8-fold, respectively, for the reduced heme iron form of the enzyme. These data support interactive binding of L-arginine and tetrahydrobiopterin in proximity to the inducible NOS heme group, as observed in the crystal structure of this enzyme. In contrast, addition of L-arginine, tetrahydrobiopterin, or both to neuronal NOS oxygenase do not markedly change its heme iron midpoint potential, with observed shifts of +19,-18, and-10 mV, respectively. These data explain the contrasting reactivities between the two NOS isoforms regarding their different NADPH consumption rates and capacity to support heme iron reduction and are indicative of the regulatory mechanisms that each enzyme employs toward electron transfer. We also examine the effects of three substrate-based inhibitors of NOS on the heme iron midpoint potentials. Among these inhibitors, S-ethylisothiourea decreased the heme potentials of tetrahydrobiopterin-bound inducible NOS and neuronal NOS by 27 and 24 mV, respectively, N-nitro-L-arginine methyl ester lowered both potentials below-460 mV, and aminoguanidine slightly increased both potentials. This work suggests the following: (1) A thermodynamic block of reductase-catalyzed heme reduction exists in inducible NOS but not in neuronal NOS in the absence of substrate and tetrahydrobiopterin. This reveals distinct heme environments for the two isoforms. (2) Heme iron reduction thermodynamics in inducible NOS are improved by tetrahydrobiopterin and L-arginine, implying that this isoform is uniquely configured to respond to substrate and pterin control. (3) Some, but not all, inhibitors that reduce electron flux through NOS act by affecting the thermodynamics of heme iron reduction.
Heme coordination of NO in NO synthase
Proceedings of the National Academy of Sciences of the United States of America, 1994
A current question in nitric oxide (NO) biology is whether NO can act as a feedback inhibitor of NO synthase (NOS). We have approached this problem by examning the interaction of NO with neuronal NOS by optical absorption and resonance Raman scattering spectroscopies. Under an inert atmosphere NO coordinated to the heme Iron in both the oxidized and reduced forms of NOS. The Soret and visible optical absorption transitions are detected at 436 and at 567 nm, respectively, in the Fe2+-NO heme complex and at 440 nm and at 549 and 580 nm, respectively, in the Fe3+-NO heme complex. In the resonance Raman spectrum of the ferrous complex the Fe-NO stretching mode is located at 549 cm' in the presence of L-arglle and at 536 cm-' in the absence of L-argnine, whereas in the ferric enzyme the mode is located at 540 cm-(in the absence of L-anne). The interaction between bound L-argine and the NO indicates that L-argine binds directly over the heme just as do the substrates in cytochrome P-450s. In the absence of L-argnine, NO readily oxidized the ferrous heme Iron. The oxidation was prevented by the presence of bound L-arglnine and enabled NOS to form a stable ferrous NO complex. Under oxygen-limited conditions, NO generated by neuronal NOS coordinated to Its heme iron and formed a spectrally detectable ferrous-NO complex. Taken together, our results show that NO can bind to both ferric and ferrous NOS and may inhibit NO synthesis through its binding to the heme iron during catalysis.