L-Arg Binding Research Articles

Gelation improvement of low-salt Chinese shrimp (Fenneropenaeus chinensis) surimi gel by L-arginine

In this study, the effect of L-arginine (L-Arg) on the gel properties of low-salt (NaCl, 0.5 % w/w) Chinese shrimp surimi gel (SSG) was investigated and the mechanism was explored. As the L-Arg concentration was raised from 0 to 0.75 %, the gel strength and hardness of SSG reached the maximum values and remained stable, increasing by 7.63 % and 82.5 %, respectively, and these results were better than those of high-salt (2 % NaCl) control group. Moreover, L-Arg enhanced the apparent viscosity of minced shrimp, decreased the G' of minced shrimp but increased the G' of SSG. With the addition of L-Arg, the protein solubility was significantly elevated to 74.89 %. The binding of L-Arg to Chinese shrimp myosin was mainly dependent on hydrogen bonds. In summary, 0.75 % L-Arg increased protein solubility, hydrogen bonds and disulfide bonds, forming a denser gel network structure for low-salt SSG, thus improving gel properties.

GPRC6A is a Potential Therapeutic Target for Metformin Regulation of Glucose Homeostasis in Mice.

Understanding the mechanism of metformin actions in treating type 2 diabetes is limited by an incomplete knowledge of the specific protein targets mediating its metabolic effects. Metformin has structural similarities to L-Arginine (2-amino-5-guanidinopentanoic acid), which is a ligand for GPRC6A, a Family C G-protein coupled receptor that regulates energy metabolism. Ligand activation of GPRC6A results in lowering of blood glucose and other metabolic changes resembling the therapeutic effect of metformin. In the current study, we tested if metformin activates GPRC6A. We used Alphafold2 to develop a structural model for L-Arginine (L-Arg) binding to the extracellu-lar bilobed venus flytrap domain (VFT) of GPRC6A. We found that metformin docked to the site in the VFT that overlaps the binding site for L-Arg. Metformin resulted in a dose-dependent stimulation of GPRC6A activity in HEK-293 cells transfected with full-length wild-type GPRC6A but not in untransfected control cells. In addition, metformin failed to activate an alternatively spliced GPRC6A isoform lacking the putative binding site in the VFT. More specifically, mutation of the predicted metformin key binding residues Glu170 and Asp303 in the GPRC6A VFT resulted in loss of metformin receptor activation in vitro. The in vivo role of GPRC6A in mediating the effects of metformin was tested in Gprc6a-/- mice. Administration of therapeutic doses of metformin lowered blood glucose levels following a glucose tolerance test in wild-type but not Gprc6a-/- mice. Finally, we EN300, created by adding a carboxymethyl group from L-Arg to the biguanide backbone of metformin. EN300 showed dose-dependent stimulation of GPRC6A activity in vitro with greater potency than L-Arginine, but less than metformin. Thus, we suggest that GPRC6A is a potential molecular target for metformin which may be used to understand the therapeutic actions of metformin and develop novel small molecules to treat T2D.

Novel synergistic effect-based fluorescent sensor for highly selective and sensitive detection of l-Arg

Since l-arginine (l-Arg) is crucial to the biological system, it is important to detect l-Arg effectively. Here, we created and synthesized a brand-new fluorescence sensor (FAS) to detect l-Arg with greater sensitivity. Four acylhyhydrazone functionalized arms and two hydroxyl groups have been logically added to the FAS to increase the selectivity and sensitivity of the l-Arg sensor. These groups work together to provide effective l-Arg binding. The findings demonstrated that the FAS could specifically bind l-Arg by collaborating with the tetra-acylhyhydrazone and two hydroxyl groups through multiple intermolecular hydrogen bonds and electrostatic attractions; the sensitivity of the FAS to detect l-Arg is high, with a detection limit of 2.73 × 10−8 mol/L. Theoretical calculations and tests were used to study the potential recognition process. Furthermore, the chemosensor FAS was effectively used to ascertain the l-Arg concentration in human blood samples, indicating its capability to identify l-Arg in diverse biological matrices.

Structural, Spectroscopic, and Computational Insights from Canavanine-Bound and Two Catalytically Compromised Variants of the Ethylene-Forming Enzyme.

The ethylene-forming enzyme (EFE) is an Fe(II), 2-oxoglutarate (2OG), and l-arginine (l-Arg)-dependent oxygenase that either forms ethylene and three CO2/bicarbonate from 2OG or couples the decarboxylation of 2OG to C5 hydroxylation of l-Arg. l-Arg binds with C5 toward the metal center, causing 2OG to change from monodentate to chelate metal interaction and OD1 to OD2 switch of D191 metal coordination. We applied anaerobic UV-visible spectroscopy, X-ray crystallography, and computational approaches to three EFE systems with high-resolution structures. The ineffective l-Arg analogue l-canavanine binds to the EFE with O5 pointing away from the metal center while promoting chelate formation by 2OG but fails to switch the D191 metal coordination from OD1 to OD2. Substituting alanine for R171 that interacts with 2OG and l-Arg inactivates the protein, prevents metal chelation by 2OG, and weakens l-Arg binding. The R171A EFE had electron density at the 2OG binding site that was identified by mass spectrometry as benzoic acid. The substitution by alanine of Y306 in the EFE, a residue 12 Å away from the catalytic metal center, generates an interior cavity that leads to multiple local and distal structural changes that reduce l-Arg binding and significantly reduce the enzyme activity. Flexibility analyses revealed correlated and anticorrelated motions in each system, with important distinctions from the wild-type enzyme. In combination, the results are congruent with the currently proposed enzyme mechanism, reinforce the importance of metal coordination by OD2 of D191, and highlight the importance of the second coordination sphere and longer range interactions in promoting EFE activity.

Small chemical compounds Y16 and Rhosin can inhibit calcium sensitization pathway in vascular smooth muscle cells of spontaneously hypertensive rats

The small-molecule compounds Y16 and Rhosin can inhibit the activation of leukemia-associated Rho guanine nucleotide exchange factor (LARG) and small G-protein RhoA, respectively, in breast cancer cells and inhibit their growth and migration. However, it remains unclear whether they have inhibitory effects on the vascular smooth muscle cells (VSMCs) of spontaneously hypertensive rats (SHRs). Primary cultured VSMCs from SHRs were treated with different concentrations of Y16 or Y16 plus Rhosin for 24h, followed by 10-min stimulation with 10-7M angiotensin II (Ang II). The cells were then harvested, and the total protein was extracted. The co-immunoprecipitation method, Western blot analysis, and MTT assay were performed to determine the LARG-RhoA interaction, the protein levels of RhoA and MYPT1, and cell viability, respectively. Y16 dose-dependently inhibited the LARG-RhoA complex formation induced by Ang II. With 50μM of Y16, the effect of inhibition was statistically significant. Y16 also reduced the formation of phospho-MYPT1 stimulated by Ang II. With 5μM of Y16, the inhibitory effect was statistically significant. When 25μM of Y16 and 25μM of Rhosin were combined, the inhibitory effect on LARG-RhoA interaction was statistically significant. When Y16 and Rhosin were combined, a significantly reduced concentration could effectively inhibit MYPT1 phosphorylation (2.5μM compared with 5μM for Y16 alone). Treating SHR VSMCs with Y16 can suppress the activation of LARG, prevent LARG binding to RhoA, and decrease the phosphorylation of MYPT1, thus weakening the activation of the calcium (Ca2+) sensitization pathway in SHR VSMCs.

Conserved Dynamic Mechanism of Allosteric Response to L-arg in Divergent Bacterial Arginine Repressors.

Hexameric arginine repressor, ArgR, is the feedback regulator of bacterial L-arginine regulons, and sensor of L-arg that controls transcription of genes for its synthesis and catabolism. Although ArgR function, as well as its secondary, tertiary, and quaternary structures, is essentially the same in E. coli and B. subtilis, the two proteins differ significantly in sequence, including residues implicated in the response to L-arg. Molecular dynamics simulations are used here to evaluate the behavior of intact B. subtilis ArgR with and without L-arg, and are compared with prior MD results for a domain fragment of E. coli ArgR. Relative to its crystal structure, B. subtilis ArgR in absence of L-arg undergoes a large-scale rotational shift of its trimeric subassemblies that is very similar to that observed in the E. coli protein, but the residues driving rotation have distinct secondary and tertiary structural locations, and a key residue that drives rotation in E. coli is missing in B. subtilis. The similarity of trimer rotation despite different driving residues suggests that a rotational shift between trimers is integral to ArgR function. This conclusion is supported by phylogenetic analysis of distant ArgR homologs reported here that indicates at least three major groups characterized by distinct sequence motifs but predicted to undergo a common rotational transition. The dynamic consequences of L-arg binding for transcriptional activation of intact ArgR are evaluated here for the first time in two-microsecond simulations of B. subtilis ArgR. L-arg binding to intact B. subtilis ArgR causes a significant further shift in the angle of rotation between trimers that causes the N-terminal DNA-binding domains lose their interactions with the C-terminal domains, and is likely the first step toward adopting DNA-binding-competent conformations. The results aid interpretation of crystal structures of ArgR and ArgR-DNA complexes.

A Kinetic Study of Ligand Binding and Conformational Changes in Inducible Nitric Oxide Synthase

The mammalian inducible nitric oxide synthase (iNOS) catalyzes the production of nitric oxide (NO) in a two-step monooxygenation of L-arginine (L-Arg) to NO and L-citrulline via the intermediate Nω-hydroxy-L-arginine (NOHA). The reaction starts with the formation of a ternary complex consisting of the enzyme, an O2 ligand bound to the heme iron and the L-Arg substrate residing close to the heme-bound ligand. We have blocked the enzymatic turnover by replacing O2 by carbon monoxide (CO), because it resembles O2 in size and heme binding properties, and then studied CO binding to the iNOS oxygenase domain, iNOSoxy, by using flash photolysis. The P420 and P450 forms of the enzyme, assigned to a protonated and unprotonated proximal cysteine, through which the heme is anchored to the protein, show markedly different CO rebinding properties. Only very few CO ligands can escape from P420. They rebind very slowly, suggesting that P420 has a widely open distal pocket that admits water. CO rebinding to the P450 form strongly depends on the presence of the substrate L-Arg, the intermediate Nω-hydroxy-L-arginine and also the cofactor tetrahydrobiopterin. After adding these small molecules to the enzyme solution, the CO kinetics change slowly over hours. This process can be described as a relaxation from a fast rebinding, metastable P450 species to a slowly rebinding, thermodynamically stable P450 species, which we associate with the enzymatically active form. Our results allow us to determine kinetic parameters of L-Arg binding to ferrous deoxy iNOS protein, the L-Arg dissociation rate coefficient,= 15.4 ± 0.4 s−1, the bimolecular L-Arg association rate coefficient, = 1153 ± 71 mM−1s−1, and by taking the ratio of the two values, the equilibrium dissociation coefficient of L-Arg and ferrous iNOSoxy, KD = 13.4 ± 0.9 μM.

Kinetic Study of Ligand Binding and Conformational Changes in Inducible Nitric Oxide Synthase.

Nitric oxide synthases (NOSs) are heme enzymes that generate highly reactive nitric oxide from l-arginine (l-Arg) in a complex mechanism that is still only partially understood. We have studied carbon monoxide (CO) binding to the oxygenase domain of murine inducible NOS (iNOS) by using flash photolysis. The P420 and P450 forms of the enzyme, assigned to a protonated and unprotonated proximal cysteine, through which the heme is anchored to the protein, show markedly different CO rebinding properties. The data suggest that P420 has a widely open distal pocket that admits water. CO rebinding to the P450 form strongly depends on the presence of the substrate l-Arg, the intermediate Nω-hydroxy-l-arginine, and the cofactor tetrahydrobiopterin. After adding these small molecules to the enzyme solution, the CO kinetics change slowly over the hours. This process can be described as a relaxation from a fast rebinding, metastable species to a slowly rebinding, thermodynamically stable species, which we associate with the enzymatically active form. Our results allow us to determine kinetic parameters of l-Arg binding to the ferrous deoxy iNOS protein for the first time and also provide clues regarding the nature of structural differences between the two interconverting species.

Structures and Mechanisms of the Non-Heme Fe(II)- and 2-Oxoglutarate-Dependent Ethylene-Forming Enzyme: Substrate Binding Creates a Twist.

The ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2 is a member of the mononuclear nonheme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenase superfamily. EFE converts 2OG into ethylene plus three CO2 molecules while also catalyzing the C5 hydroxylation of l-arginine (l-Arg) driven by the oxidative decarboxylation of 2OG to form succinate and CO2. Here we report 11 X-ray crystal structures of EFE that provide insight into the mechanisms of these two reactions. Binding of 2OG in the absence of l-Arg resulted in predominantly monodentate metal coordination, distinct from the typical bidentate metal-binding species observed in other family members. Subsequent addition of l-Arg resulted in compression of the active site, a conformational change of the carboxylate side chain metal ligand to allow for hydrogen bonding with the substrate, and creation of a twisted peptide bond involving this carboxylate and the following tyrosine residue. A reconfiguration of 2OG achieves bidentate metal coordination. The dioxygen binding site is located on the metal face opposite to that facing l-Arg, thus requiring reorientation of the generated ferryl species to catalyze l-Arg hydroxylation. Notably, a phenylalanyl side chain pointing toward the metal may hinder such a ferryl flip and promote ethylene formation. Extensive site-directed mutagenesis studies supported the importance of this phenylalanine and confirmed the essential residues used for substrate binding and catalysis. The structural and functional characterization described here suggests that conversion of 2OG to ethylene, atypical among Fe(II)/2OG oxygenases, is facilitated by the binding of l-Arg which leads to an altered positioning of the carboxylate metal ligand, a resulting twisted peptide bond, and the off-line geometry for dioxygen coordination.

Small-molecule inhibitors targeting G-protein–coupled Rho guanine nucleotide exchange factors

The G-protein–mediated Rho guanine nucleotide exchange factor (GEF)–Rho GTPase signaling axis has been implicated in human pathophysiology and is a potential therapeutic target. By virtual screening of chemicals that fit into a surface groove of the DH-PH domain of LARG, a G-protein–regulated Rho GEF involved in RhoA activation, and subsequent validations in biochemical assays, we have identified a class of chemical inhibitors represented by Y16 that are active in specifically inhibiting LARG binding to RhoA. Y16 binds to the junction site of the DH-PH domains of LARG with a ∼80 nM K d and suppresses LARG catalyzed RhoA activation dose dependently. It is active in blocking the interaction of LARG and related G-protein–coupled Rho GEFs with RhoA without a detectable effect on other DBL family Rho GEFs, Rho effectors, or a RhoGAP. In cells, Y16 selectively inhibits serum-induced RhoA activity and RhoA-mediated signaling, effects that can be rescued by a constitutively active RhoA or ROCK mutant. By suppressing RhoA activity, Y16 inhibits mammary sphere formation of MCF7 breast cancer cells but does not affect the nontransforming MCF10A cells. Significantly, Y16 works synergistically with Rhosin/G04, a Rho GTPase activation site inhibitor, in inhibiting LARG–RhoA interaction, RhoA activation, and RhoA-mediated signaling functions. Thus, our studies show that Rho GEFs can serve as selective targets of small chemicals and present a strategy of dual inhibition of the enzyme–substrate pair of GEF–RhoA at their binding interface that leads to enhanced efficacy and specificity.

L-Arginine Binding to Human Inducible Nitric Oxide Synthase: An Antisymmetric Funnel Route toward Isoform-Specific Inhibitors?

Nitric oxide (NO) is an important signaling molecule produced by a family of enzymes called nitric oxide synthases (NOS). Because NO is involved in various pathological conditions, the development of potent and isoform-selective NOS inhibitors is an important challenge. In the present study, the dimer of oxygenase domain of human iNOS (iNOSoxy) complexed to its natural substrate L-arginine (L-Arg) and both heme and tetrahydro-L-biopterin (BH4) cofactors was studied through multiple molecular dynamics simulations. Starting from the X-ray structure available for that complex (PDB: 1NSI ), a 16 ns equilibration trajectory was first obtained. Twelve dynamics of slow extraction of L-Arg out from the iNOSoxy active site were then performed. The steered molecular dynamics (SMD) approach was used starting from three different points of the reference trajectory for a total simulation time of 35 ns. A probable unbinding/binding pathway of L-Arg was characterized. It was suggested that a driving force directed the substrate toward the heme pocket. Key intermediate steps/residues along the access route to the active site were identified along this "funnel shape" pathway and compared to existing data. A quasi-normal mode analysis performed on the SMD data suggested that large collective motions of the protein may be involved in L-Arg binding and that opening the route to the active site in one monomer promoted an inverse, closing motion in the second monomer. Finally, our findings might help to rationalize the design of human iNOS isoform competitive inhibitors.

Stabilization of Reaction Intermediates in the Catalytic Cycle of Nitric Oxide Synthases

Nitric oxide synthases (NOS) catalyze the synthesis of nitric oxide from L‐Arginine. NOS enzymes have a tryptophan residue that forms a hydrogen bond with the heme‐thiolate and a stacking interaction with the heme. To assess the importance of this residue we replaced it by histidine in inducible NOS. The W188H mutation had relatively small effects on L‐Arg binding and on enzyme heme‐CO and heme‐NO absorbance spectra, but increased the heme midpoint potential by 88 mV relative to wild‐type iNOSoxy. The crystal structure of the mutant showed that the His188 imidazole still stacked with the heme and was in position to form a hydrogen bond with the heme thiolate. Analysis of a single turnover L‐Arg hydroxylation reaction revealed that a new heme species formed during the reaction. Formation of this species required the presence of tetrahydrobiopterin (H4B) and its buildup appears to be coupled with the formation of the bound H4B radical. The decay of the intermediate species correlates with L‐Arg hydroxylation and formation of ferric enzyme.This indicates that the W188H mutation stabilizes an intermediate heme‐oxy species whose reactivity becomes rate‐limiting for L‐Arg hydroxylation. The conserved Trp residue in NOS may facilitate NO synthesis by tuning heme‐thiolate electronegativity. Additional research to identify the intermediate will be discussed.

Stabilization and Characterization of a Heme-Oxy Reaction Intermediate in Inducible Nitric-oxide Synthase

Nitric-oxide synthases (NOS) are heme-thiolate enzymes that N-hydroxylate L-arginine (L-Arg) to make NO. NOS contain a unique Trp residue whose side chain stacks with the heme and hydrogen bonds with the heme thiolate. To understand its importance we substituted His for Trp188 in the inducible NOS oxygenase domain (iNOSoxy) and characterized enzyme spectral, thermodynamic, structural, kinetic, and catalytic properties. The W188H mutation had relatively small effects on l-Arg binding and on enzyme heme-CO and heme-NO absorbance spectra, but increased the heme midpoint potential by 88 mV relative to wild-type iNOSoxy, indicating it decreased heme-thiolate electronegativity. The protein crystal structure showed that the His188 imidazole still stacked with the heme and was positioned to hydrogen bond with the heme thiolate. Analysis of a single turnover L-Arg hydroxylation reaction revealed that a new heme species formed during the reaction. Its build up coincided kinetically with the disappearance of the enzyme heme-dioxy species and with the formation of a tetrahydrobiopterin (H4B) radical in the enzyme, whereas its subsequent disappearance coincided with the rate of l-Arg hydroxylation and formation of ferric enzyme. We conclude: (i) W188H iNOSoxy stabilizes a heme-oxy species that forms upon reduction of the heme-dioxy species by H4B. (ii) The W188H mutation hinders either the processing or reactivity of the heme-oxy species and makes these steps become rate-limiting for l-Arg hydroxylation. Thus, the conserved Trp residue in NOS may facilitate formation and/or reactivity of the ultimate hydroxylating species by tuning heme-thiolate electronegativity.

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.

Thermodynamic analysis of l-arginine and Nω-hydroxy- l-arginine binding to nitric oxide synthase

Isothermal titration calorimetry has been used to determine thermodynamic parameters of substrate binding to the oxygenase domain of neuronal nitric oxide synthase (nNOS oxy) in the presence of the cofactor tetrahydrobiopterin. The intermediate N ω-hydroxy- l-arginine (NHA) has a larger affinity than l-Arginine ( l-Arg) for nNOS oxy, with K d = 0.4 ± 0.1 µM and 1.7 ± 0.3 µM at 25 °C, respectively. nNOS oxy binds NHA and l-Arg with Δ H − 4.1 ± 0.2 and − 1.0 ± 0.1 kcal/mol and Δ S = 15 and 23 cal/K mol respectively. NHA binding is more exothermic probably due to formation of an extra hydrogen bond in the active site compared to l-Arg. The changes in heat capacity (Δ C p) are relatively small for binding of both NHA and l-Arg (− 53 ± 18 and − 95 ± 23 cal/L mol, respectively), which indicates that hydrophobic interactions contribute little to binding.

Pharmacologic Manipulation of Nitric Oxide Signaling: Targeting NOS Dimerization and Protein-Protein Interactions

Nitric oxide (NO) is an endogenously-produced small molecule that has critical roles in cellular signaling and a variety of physiological processes in many tissues, including the brain, the vasculature, and the immune system. In several medical disorders, NO has been implicated in disease pathology, in most cases due to persistent activation or overproduction of one of three NO synthase (NOS) isoforms. Although NOS inhibitors that are both potent and cell-permeable have been developed, none is currently used in the treatment of any disorder. One reason that NOS inhibitors fail to have therapeutic efficacy may be linked to their very low isoform-selectivity. An additional possibility is that NOS inhibitors, even if they exhibit isoform selectivity, might indiscriminately affect beneficial and pathological NO signaling pathways. In this review, we discuss emerging approaches in the development of isoform-specific NOS-directed therapeutics including dimerization inhibitors, novel L-arginine (L-Arg) binding site inhibitors, and dimer stabilization. Additionally, we suggest novel strategies for the future including targeting subcellular localization of NOS and protein-protein interactions with NOS effectors.

Developmental differences in pulmonary nitric oxide (NO) production and endogenous nitric oxide synthase inhibitors in the neonatal rat

We recently observed differences in exhaled NO production in isolated perfused lungs in 7 and 14 d/o rats, where 7 d lungs produced less than 14 d lungs. These differences were independent of NO synthase (NOS) protein levels. Therefore, mechanisms responsible for NOS activation and inhibition were evaluated. We tested the hypothesis that the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) plays a role in modulating NOS activity in the developing pulmonary vasculature. Whole lung homogenates and plasma were obtained from 7 and 14 d/o rats and snap frozen. The homogenates were then compared by immunoblot analysis for site specific eNOS phosphorylation and compared by immune-mediated co-precipitation experiments to assess eNOS associated HSP90 or Caveolin 1 (Cav 1) levels. Finally, we used HPLC to measure plasma ADMA and arginine (L-Arg) levels. We found no differences in eNOS associated HSP90 or Cav 1 protein levels, nor were there differences in eNOS phosphorylation profiles. However, plasma ADMA levels were 2 fold greater in the 7 vs. 14 d/o rats and the plasma L-Arg/ADMA ratio was shifted lower in the 7 vs. 14 d/o rats. The shift in the L-Arg/ADMA ratio is significant because NOS uses LArg as its substrate and ADMA is a competitive inhibitor of L-Arg binding. This ratio allows one to predict the degree of NOS inhibition. Based on previously published Ki’s for NOS, we expect that the decreased ratio observed could inhibit NOS by as much as 30–40%, which may result in impaired endothelial function in the early neonatal course.

Regulation of the Monomer-Dimer Equilibrium in Inducible Nitric-oxide Synthase by Nitric Oxide

The oxygenase domain of inducible nitric-oxide synthase exists as a functional tight homodimer in the presence of the substrate L-arginine and the cofactor tetrahydrobiopterin (H4B). In the absence of H4B, the enzyme is a mixture of monomer and loose dimer. We show that exposure of H4B-free enzyme to NO induces dissociation of the loose dimer into monomers in a reaction that follows single exponential decay kinetics with a lifetime of approximately 300 min. It is followed by a faster autoreduction reaction of the heme iron with a lifetime of approximately 30 min and the concurrent breakage of the proximal iron-thiolate bond, forming a five-coordinate NO-bound ferrous species. Mass spectrometry revealed that the NO-induced monomerization is associated with intramolecular disulfide bond formation between Cys104 and Cys109, located in the zinc-binding motif. The regulatory effect of NO as a dimer inhibitor is discussed in the context of the structure/function relationships of this enzyme.

Nitric-oxide Synthase Output State

Mammalian nitric-oxide synthases are large modular enzymes that evolved from independently expressed ancestors. Calmodulin-controlled isoforms are signal generators; calmodulin activates electron transfer from NADPH through three reductase domains to an oxygenase domain. Structures of the reductase unit and its homologs show FMN and FAD in contact but too isolated from the protein surface to permit exit of reducing equivalents. To study states in which FMN/heme electron transfer is feasible, we designed and produced constructs including only oxygenase and FMN binding domains, eliminating strong internal reductase complex interactions. Constructs for all mammalian isoforms were expressed and purified as dimers. All synthesize NO with peroxide as the electron donor at rates comparable with corresponding oxygenase constructs. All bind cofactors nearly stoichiometrically and have native catalytic sites by spectroscopic criteria. Modest differences in electrochemistry versus independently expressed heme and FMN binding domains suggest interdomain interactions. These interactions can be convincingly demonstrated via calmodulin-induced shifts in high spin ferriheme EPR spectra and through mutual broadening of heme and FMNH. radical signals in inducible nitric-oxide synthase constructs. Blue neutral FMN semiquinone can be readily observed; potentials of one electron couple (in inducible nitric-oxide synthase oxygenase FMN, FMN oxidized/semiquinone couple = +70 mV, FMN semiquinone/hydroquinone couple = -180 mV, and heme = -180 mV) indicate that FMN is capable of serving as a one electron heme reductant. The construct will serve as the basis for future studies of the output state for NADPH derived reducing equivalents.

Heme Distortion Modulated by Ligand-Protein Interactions in Inducible Nitric-oxide Synthase

The catalytic center of nitric-oxide synthase (NOS) consists of a thiolate-coordinated heme macrocycle, a tetrahydrobiopterin (H4B) cofactor, and an l-arginine (l-Arg)/N-hydroxyarginine substrate binding site. To determine how the interplay between the cofactor, the substrates, and the protein matrix housing the heme regulates the enzymatic activity of NOS, the CO-, NO-, and CN(-)-bound adducts of the oxygenase domain of the inducible isoform of NOS (iNOS(oxy)) were examined with resonance Raman spectroscopy. The Raman data of the CO-bound ferrous protein demonstrated that the presence of l-Arg causes the Fe-C-O moiety to adopt a bent structure because of an H-bonding interaction whereas H4B binding exerts no effect. Similar behavior was found in the CN(-)-bound ferric protein and in the nitric oxide (NO)-bound ferrous protein. In contrast, in the NO-bound ferric complexes, the addition of l-Arg alone does not affect the structural properties of the Fe-N-O moiety, but H4B binding forces it to adopt a bent structure, which is further enhanced by the subsequent addition of l-Arg. The differential interactions between the various heme ligands and the protein matrix in response to l-Arg and/or H4B binding is coupled to heme distortions, as reflected by the development of a variety of out-of-plane heme modes in the low frequency Raman spectra. The extent and symmetry of heme deformation modulated by ligand, substrate, and cofactor binding may provide important control over the catalytic and autoinhibitory properties of the enzyme.