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1.
Biophys Chem ; 262: 106379, 2020 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-32339785

RESUMO

Reactive oxygen species (ROS) produced by NADPH oxidase 5 (Nox5) are regulated by Ca2+ flux through the interactions of its self-contained EF-hand domain (EFD), dehydrogenase domain (DH), and transmembrane domain. Studies suggest that the regulatory EF-hand binding domain (REFBD) and phosphorylatable (PhosR) sequences within DH play an important role in Nox5's superoxide-generating activity. However, the interplay of the EFD-DH interaction is largely unclear. Here, we used two synthetic peptides corresponding to the putative REFBD and PhosR sequences, as well as DH construct proteins, and separately studied their binding to EFD by fluorescence spectroscopy and calorimetry. With mutagenesis, we revealed that the C-terminal half domain of EFD binds specifically to REFBD in a Ca2+-dependent manner, which is driven primarily by hydrophobic interactions to form a more compact structure. On the other hand, the interaction between EFD and PhosR is not Ca2+-dependent and is primarily dominated by electrostatic interactions. The binding constants (Ka) for both peptides to EFD were calculated to be in the range of 105 M-1. The formation of the binary complex EFD/REFBD and ternary complex EFD/REFBD/PhosR was demonstrated by fluorescence resonance energy transfer (FRET). However, EFD binding to PhosR appears to be not biologically important while the conformational change on its C-terminal half domain resembles a major factor in EFD-DH domain-domain interactions.

2.
J Biomol Struct Dyn ; : 1-17, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31203730

RESUMO

The superoxide-generating activity of Nox5 is regulated by Ca2+ flux, primarily through its self-contained calcium binding domain (EFD). Upon Ca2+ binding, Nox5's EFD undergoes a conformational change that exposes its buried hydrophobic residues. Previously, we determined the Ca2+ binding constants of the N-terminal half domain (N-EFD). Here we performed a similar characterization with its C-terminal lobe (C-EFD). Our studies revealed that the binding affinities (Ka's) of the EFD are in the range of 108-105 M-1 with a strong Ca2+ binding that occurs in the C-EFD. The 3rd Ca2+ binding site also binds Mg2+ (Ka = 4.53 × 103 M-1), where its high Ca2+ binding affinity becomes moderate in cellular conditions. The essential hydrophobic exposure upon metal binding was assessed with the analysis of the 1-anilino-8-naphthalene sulfonate (ANS) interaction via fluorescence and calorimetry. While the ANS fluorescence and binding studies agree with each other in general, the results do not correlate to the actual hydrophobic exposure content. The heat capacity change (ΔCp) of Ca2+ binding for EFD is -24.1 cal/mol.K, while those of N-EFD and C-EFD are -56.3 and -41.6 cal/mol·K, respectively, indicating a significant hydrophobic exposure and polar burial. The latter was confirmed by limited trypsin digestion. The comparison of Nox5's EFD to calmodulin, including homology modeling, was discussed in the report. Communicated by Ramaswamy H. Sarma.

3.
Acta Biomater ; 90: 424-440, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30953801

RESUMO

During development of mineralized collagenous tissues, intrafibrillar mineralization is achieved by preventing mineralization precursor inhibitors that are larger than 40 kDa from entering the collagen fibrils. Such a property is incorporated in the design of a calcium chelator for dentin bonding in the etch-and-rinse technique that selectively demineralizes extrafibrillar apatite while leaving the intrafibrillar minerals intact. This strategy prevents complete demineralization of collagen fibrils, avoids collapse of collagen that blocks resin infiltration after air-drying, and protects the completely demineralized fibrils from bacteria colonization and degradation by endogenous proteases after resin bonding. In the present study, a water-soluble glycol chitosan-EDTA (GCE) conditioner was synthesized by conjugation of EDTA, an effective calcium chelator, to high molecular weight glycol chitosan, which exhibits weak chelation property. The GCE conjugate was purified, characterized by FTIR, 1H NMR, isothermal titration calorimetry and ICP-AES, and subjected to size exclusion dialysis to recover molecules that are >40 kDa. The optimal concentration and application time for etching dentin were determined by bond strength testing to ensure that the dentin bonding results were comparable to phosphoric acid etching, and maintained equivalent bond strength after air-drying of the conditioned collagen matrix. Extrafibrillar demineralization was validated with transmission electron microscopy. Inhibition of endogenous dentin proteases was confirmed using in-situ zymography. The water-soluble GCE dentin conditioner was non-cytotoxic and possessed antibacterial activities against planktonic and single-species biofilms, supporting its ongoing development as a dentin conditioner with air-drying, anti-proteolytic and antibacterial properties to enhance the durability of bonds created using the etch-and-rinse bonding technique. STATEMENT OF SIGNIFICANCE: The current state-of-the-art techniques for filling decayed teeth with plastic tooth-colored materials require conditioning the mineralized, biofilm-covered, decayed dentin with acids or acid resin monomers to create a surface layer of completely- or partially-demineralized collagen matrix for the infiltration of adhesive resin monomers. Nevertheless, fillings prepared using these strategies are not as durable as consumers have anticipated. Conjugation of polymeric glycol chitosan with EDTA produces a new conditioner for dentin bonding that demineralizes only extrafibrillar dentin, reduces endogenous protease activities and kills biofilm bacteria. The high molecular weight glycol chitosan-EDTA is non-cytotoxic to the key regenerative players within the dentin-pulp complex. This advance permits dry bonding and the use of hydrophobic resins.

4.
Acta Biomater ; 57: 435-448, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28499631

RESUMO

Limitations associated with wet-bonding led to the recent development of a selective demineralization strategy in which dentin was etched with a reduced concentration of phosphoric acid to create exclusive extrafibrillar demineralization of the collagen matrix. However, the use of acidic conditioners removes calcium via diffusion of very small hydronium ions into the intrafibrillar collagen water compartments. This defeats the purpose of limiting the conditioner to the extrafibrillar space to create a collagen matrix containing only intrafibrillar minerals to prevent collapse of the collagen matrix. The present work examined the use of polymeric chelators (the sodium salt of polyacrylic acid) of different molecular weights to selectively demineralize extrafibrillar dentin. These polymeric chelators exhibit different affinities for calcium ions (isothermal titration calorimetry), penetrated intrafibrillar dentin collagen to different extents based on their molecular sizes (modified size-exclusion chromatography), and preserve the dynamic mechanical properties of mineralized dentin more favorably compared with completely demineralized phosphoric acid-etched dentin (nanoscopical dynamic mechanical analysis). Scanning and transmission electron microscopy provided evidence for retention of intrafibrillar minerals in dentin surfaces conditioned with polymeric chelators. Microtensile bond strengths to wet-bonded and dry-bonded dentin conditioned with these polymeric chelators showed that the use of sodium salts of polyacrylic acid for chelating dentin prior to bonding did not result in significant decline in resin-dentin bond strength. Taken together, the findings led to the conclusion that a chelate-and-rinse conditioning technique based on extrafibrillar collagen demineralization bridges the gap between wet and dry dentin bonding. STATEMENT OF SIGNIFICANCE: The chelate-and-rinse dental adhesive bonding concept differentiates from previous research in that it is based on the size-exclusion characteristics of fibrillar collagen; molecules larger than 40kDa are prevented from accessing the intrafibrillar water compartments of the collagen fibrils. Using this chelate-and-rinse extrafibrillar calcium chelation concept, collagen fibrils with retained intrafibrillar minerals will not collapse upon air-drying. This enables adhesive infiltration into the mineral-depleted extrafibrillar spaces without relying on wet-bonding. By bridging the gap between wet and dry dentine bonding, the chelate-and-rinse concept introduces additional insight to the field by preventing exposure of endogenous proteases via preservation of the intrafibrillar minerals within a collagen matrix. If successfully validated, this should help prevent degradation of resin-dentine bonds by collagenolytic enzymes.


Assuntos
Colágeno/química , Dentina/química , Dente Molar/química , Desmineralização do Dente , Humanos
5.
J Inorg Biochem ; 158: 122-130, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27013266

RESUMO

Nitric oxide synthases (NOSs) catalyze a two-step oxidation of l-arginine to form nitric oxide (NO) and l-citrulline. NOS contains a N-terminal oxygenase domain (NOSoxy) that is the site of NO synthesis, and a C-terminal reductase domain (NOSred) that binds nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN) and provides electrons to the NOSoxy heme during catalysis. The three NOS isoforms in mammals inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) share high structural similarity but differ in NO release rates and catalytic properties due to differences in enzyme kinetic parameters. These parameters must be balanced for NOS enzymes to release NO, rather than consume it in a competing, inherent NO dioxygenase reaction. To improve understanding, we drew on a global catalytic model and previous findings to design three NOS chimeras that may predominantly function as NO dioxygenases: iNOSoxy/nNOSred (Wild type (WT) chimera), V346I iNOSoxy/nNOSred (V346I chimera) and iNOSoxy/S1412D nNOSred (S1412D chimera). The WT and S1412D chimeras had higher NO release than the parent iNOS, while the V346I chimera exhibited much lower NO release, consistent with expectations. Measurements indicated that a greater NO dioxygenase activity was achieved, particularly in the V346I chimera, which dioxygenated an estimated two to four NO per NO that it released, while the other chimeras had nearly equivalent NO dioxygenase and NO release activities. Computer simulations of the global catalytic model using the measured kinetic parameters produced results that mimicked the measured outcomes, and this provided further insights on the catalytic behaviors of the chimeras and basis of their increased NO dioxygenase activities.


Assuntos
Óxido Nítrico Sintase/metabolismo , Oxigenases/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/metabolismo , Modelos Biológicos , Estrutura Molecular , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/genética , Oxigenases/química , Oxigenases/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
6.
BMC Biochem ; 16: 6, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25888318

RESUMO

BACKGROUND: Calmodulin (CaM) plays an important role in Ca(2+)-dependent signal transduction. Ca(2+) binding to CaM triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition. CaM regulates a Ca(2+)-dependent inactivation process in store-operated Ca(2+) entry, by interacting Orai1. To understand the relationship between Ca(2+)-induced hydrophobicity and CaM/Orai interaction, chimera proteins constructed by exchanging EF-hands of CaM with those of Troponin C (TnC) are used as an informative probe to better understand the functionality of each EF-hand. RESULTS: ANS was used to assess the context of the induced hydrophobic surface on CaM and chimeras upon Ca(2+) binding. The exchanged EF-hands from TnC to CaM resulted in reduced hydrophobicity compared with wild-type CaM. ANS lifetime measurements indicated that there are two types of ANS molecules with rather distinct fluorescence lifetimes, each specifically corresponding to one lobe of CaM or chimeras. Thermodynamic studies indicated the interaction between CaM and a 24-residue peptide corresponding to the CaM-binding domain of Orail1 (Orai-CMBD) is a 1:2 CaM/Orai-CMBD binding, in which each peptide binding yields a similar enthalpy change (ΔH = -5.02 ± 0.13 kcal/mol) and binding affinity (K(a) = 8.92 ± 1.03 × 10(5) M(-1)). With the exchanged EF1 and EF2, the resulting chimeras noted as CaM(1TnC) and CaM(2TnC), displayed a two sequential binding mode with a one-order weaker binding affinity and lower ΔH than that of CaM, while CaM(3TnC) and CaM(4TnC) had similar binding thermodynamics as CaM. The dissociation rate constant for CaM/Orai-CMBD was determined to be 1.41 ± 0.08 s(-1) by rapid kinetics. Stern-Volmer plots of Orai-CMBD Trp76 indicated that the residue is located in a very hydrophobic environment but becomes more solvent accessible when EF1 and EF2 were exchanged. CONCLUSIONS: Using ANS dye to assess induced hydrophobicity showed that exchanging EFs for all Ca(2+)-bound chimeras impaired ANS fluorescence and/or binding affinity, consistent with general concepts about the inadequacy of hydrophobic exposure for chimeras. However, such ANS responses exhibited no correlation with the ability to interact with Orai-CMBD. Here, the model of 1:2 binding stoichiometry of CaM/Orai-CMBD established in solution supports the already published crystal structure.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Motivos EF Hand , Interações Hidrofóbicas e Hidrofílicas , Proteínas Recombinantes de Fusão/metabolismo , Troponina C/metabolismo , Sequência de Aminoácidos , Canais de Cálcio/química , Calmodulina/química , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Solventes/química , Termodinâmica , Troponina C/química
7.
Chem Cent J ; 6(1): 29, 2012 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-22490336

RESUMO

BACKGROUND: Superoxide generated by non-phagocytic NADPH oxidases (NOXs) is of growing importance for physiology and pathobiology. The calcium binding domain (CaBD) of NOX5 contains four EF-hands, each binding one calcium ion. To better understand the metal binding properties of the 1st and 2nd EF-hands, we characterized the N-terminal half of CaBD (NCaBD) and its calcium-binding knockout mutants. RESULTS: The isothermal titration calorimetry measurement for NCaBD reveals that the calcium binding of two EF-hands are loosely associated with each other and can be treated as independent binding events. However, the Ca2+ binding studies on NCaBD(E31Q) and NCaBD(E63Q) showed their binding constants to be 6.5 × 105 and 5.0 × 102 M-1 with ΔHs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1st non-canonical EF-hand is largely enhanced by the binding of Ca2+ to the 2nd canonical EF-hand. The fluorescence quenching and CD spectra support a conformational change upon Ca2+ binding, which changes Trp residues toward a more non-polar and exposed environment and also increases its α-helix secondary structure content. All measurements exclude Mg2+-binding in NCaBD. CONCLUSIONS: We demonstrated that the 1st non-canonical EF-hand of NOX5 has very weak Ca2+ binding affinity compared with the 2nd canonical EF-hand. Both EF-hands interact with each other in a cooperative manner to enhance their Ca2+ binding affinity. Our characterization reveals that the two EF-hands in the N-terminal NOX5 are Ca2+ specific. GRAPHICAL

8.
J Inorg Biochem ; 108: 203-15, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22173094

RESUMO

NO synthase enzymes (NOS) support unique single-electron transitions of a bound H(4)B cofactor during catalysis. Previous studies showed that both the pterin structure and surrounding protein residues impact H(4)B redox function during catalysis. A conserved Arg residue (Arg375 in iNOS) forms hydrogen bonds with the H(4)B ring. In order to understand the role of this residue in modulating the function of H(4)B and overall NO synthesis of the enzyme, we generated and characterized three mutants R375D, R375K and R375N of the oxygenase domain of inducible NOS (iNOSoxy). The mutations affected the dimer stability of iNOSoxy and its binding affinity toward substrates and H(4)B to varying degrees. Optical spectra of the ferric, ferrous, ferrous dioxy, ferrous-NO, ferric-NO, and ferrous-CO forms of each mutant were similar to the wild-type. However, mutants displayed somewhat lower heme midpoint potentials and faster ferrous heme-NO complex reactivity with O(2). Unlike the wild-type protein, mutants could not oxidize NOHA to nitrite in a H(2)O(2)-driven reaction. Mutation could potentially change the ferrous dioxy decay rate, H(4)B radical formation rate, and the amount of the Arg hydroxylation during single turnover Arg hydroxylation reaction. All mutants were able to form heterodimers with the iNOS G450A full-length protein and displayed lower NO synthesis activities and uncoupled NADPH consumption. We conclude that the conserved residue Arg375 (1) regulates the tempo and extent of the electron transfer between H(4)B and ferrous dioxy species and (2) controls the reactivity of the heme-based oxidant formed after electron transfer from H(4)B during steady state NO synthesis and H(2)O(2)-driven NOHA oxidation. Thus, Arg375 modulates the redox function of H(4)B and is important in controlling the catalytic function of NOS enzymes.


Assuntos
Arginina/química , Biopterina/análogos & derivados , Óxido Nítrico Sintase Tipo II/metabolismo , Biopterina/química , Biopterina/genética , Biopterina/metabolismo , Catálise , Ligação de Hidrogênio , Mutagênese Sítio-Dirigida , Óxido Nítrico Sintase Tipo II/genética , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína
9.
Open Biochem J ; 4: 59-67, 2010 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-20648216

RESUMO

Superoxide generated by human NADPH oxidase 5 (NOX5) is of growing importance for various physiological and pathological processes. The activity of NOX5 appears to be regulated by a self-contained Ca(2+) binding domain (CaBD). Recently Bánfi et al. suggest that the conformational change of CaBD upon Ca(2+) binding is essential for domain-domain interaction and superoxide production. The authors studied its structural change using intrinsic Trp fluorescence and hydrophobic dye binding; however, their conformational study was not thorough and the kinetics of metal binding was not demonstrated. Here we generated the recombinant CaBD and an E99Q/E143Q mutant to characterize them using fluorescence spectroscopy. Ca(2+) binding to CaBD induces a conformational change that exposes hydrophobic patches and increases the quenching accessibilities of its Trp residues and AEDANS at Cys107. The circular dichroism spectra indicated no significant changes in the secondary structures of CaBD upon metal binding. Stopped-flow spectrometry revealed a fast Ca(2+) dissociation from the N-terminal half, followed by a slow Ca(2+) dissociation from the C-terminal half. Combined with a chemical stability study, we concluded that the C-terminal half of CaBD has a higher Ca(2+) binding affinity, a higher chemical stability, and a slow Ca(2+) dissociation. The Mg(2+)-bound CaBD was also investigated and the results indicate that its structure is similar to the apo form. The rate of Mg(2+) dissociation was close to that of Ca(2+) dissociation. Our data suggest that the N- and C-terminal halves of CaBD are not completely structurally independent.

10.
J Inorg Biochem ; 104(3): 349-56, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20006999

RESUMO

Nitric oxide (NO) release from nitric oxide synthases (NOSs) depends on the dissociation of a ferric heme-NO product complex (Fe(III)NO) 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(III)NO 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-O(2) 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-O(2) 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.


Assuntos
Heme/metabolismo , Óxido Nítrico Sintase Tipo II , Óxido Nítrico/metabolismo , Valina/metabolismo , Animais , Bacillus subtilis/enzimologia , Sítios de Ligação , Catálise , Simulação por Computador , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Estrutura Molecular , Mutagênese Sítio-Dirigida , NADP/metabolismo , Nitratos/metabolismo , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Oxirredução , Superóxidos/metabolismo , Valina/genética
11.
J Biol Chem ; 285(5): 3064-75, 2010 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-19948738

RESUMO

Neuronal nitric-oxide synthase (nNOS) contains a unique autoinhibitory insert (AI) in its FMN subdomain that represses nNOS reductase activities and controls the calcium sensitivity of calmodulin (CaM) binding to nNOS. How the AI does this is unclear. A conserved charged residue (Lys(842)) lies within a putative CaM binding helix in the middle of the AI. We investigated its role by substituting residues that neutralize (Ala) or reverse (Glu) the charge at Lys(842). Compared with wild type nNOS, the mutant enzymes had greater cytochrome c reductase and NADPH oxidase activities in the CaM-free state, were able to bind CaM at lower calcium concentration, and had lower rates of heme reduction and NO synthesis in one case (K842A). Moreover, stopped-flow spectrophotometric experiments with the nNOS reductase domain indicate that the CaM-free mutants had faster flavin reduction kinetics and had less shielding of their FMN subdomains compared with wild type and no longer increased their level of FMN shielding in response to NADPH binding. Thus, Lys(842) is critical for the known functions of the AI and also enables two additional functions of the AI as newly identified here: suppression of electron transfer to FMN and control of the conformational equilibrium of the nNOS reductase domain. Its effect on the conformational equilibrium probably explains suppression of catalysis by the AI.


Assuntos
Calmodulina/química , Flavinas/química , Lisina/química , Neurônios/enzimologia , Óxido Nítrico Sintase/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Redutases do Citocromo/química , Humanos , Cinética , Dados de Sequência Molecular , Mutação , NADPH Oxidases/química , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Homologia de Sequência de Aminoácidos
12.
J Biol Chem ; 283(17): 11734-42, 2008 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-18283102

RESUMO

Nitric-oxide synthases (NOS) are catalytically self-sufficient flavo-heme enzymes that generate NO from arginine (Arg) and display a novel utilization of their tetrahydrobiopterin (H(4)B) cofactor. During Arg hydroxylation, H(4)B acts as a one-electron donor and is then presumed to redox cycle (i.e. be reduced back to H(4)B) within NOS before further catalysis can proceed. Whereas H(4)B radical formation is well characterized, the subsequent presumed radical reduction has not been demonstrated, and its potential mechanisms are unknown. We investigated radical reduction during a single turnover Arg hydroxylation reaction catalyzed by neuronal NOS to document the process, determine its kinetics, and test for involvement of the NOS flavoprotein domain. We utilized a freeze-quench instrument, the biopterin analog 5-methyl-H(4)B, and a method that could separately quantify the flavin and pterin radicals that formed in NOS during the reaction. Our results establish that the NOS flavoprotein domain catalyzes reduction of the biopterin radical following Arg hydroxylation. The reduction is calmodulin-dependent and occurs at a rate that is similar to heme reduction and fast enough to explain H(4)B redox cycling in NOS. These results, in light of existing NOS crystal structures, suggest a "through-heme" mechanism may operate for H(4)B radical reduction in NOS.


Assuntos
Biopterina/análogos & derivados , Óxido Nítrico Sintase/química , Animais , Arginina/química , Biopterina/química , Catálise , Cristalografia por Raios X/métodos , Espectroscopia de Ressonância de Spin Eletrônica , Flavinas/química , Radicais Livres , Cinética , Óxido Nítrico/química , Óxido Nítrico Sintase/metabolismo , Oxirredução , Pterinas/química , Ratos
13.
J Biol Chem ; 282(4): 2196-202, 2007 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-17127770

RESUMO

Unlike animal nitric-oxide synthases (NOSs), the bacterial NOS enzymes have no attached flavoprotein domain to reduce their heme and so must rely on unknown bacterial proteins for electrons. We tested the ability of two Bacillus subtilis flavodoxins (YkuN and YkuP) to support catalysis by purified B. subtilis NOS (bsNOS). When an NADPH-utilizing bacterial flavodoxin reductase (FLDR) was added to reduce YkuP or YkuN, both supported NO synthesis from either L-arginine or N-hydroxyarginine and supported a linear nitrite accumulation over a 30-min reaction period. Rates of nitrite production were directly dependent on the ratio of YkuN or YkuP to bsNOS. However, the V/Km value for YkuN (5.2 x 10(5)) was about 20 times greater than that of YkuP (2.6 x 10(4)), indicating YkuN is more efficient in supporting bsNOS catalysis. YkuN that was either photo-reduced or prereduced by FLDR transferred an electron to the bsNOS ferric heme at rates similar to those measured for heme reduction in the animal NOSs. YkuN supported a similar NO synthesis activity by a different bacterial NOS (Deinococcus radiodurans) but not by any of the three mammalian NOS oxygenase domains nor by an insect NOS oxygenase domain. Our results establish YkuN as a kinetically competent redox partner for bsNOS and suggest that FLDR/flavodoxin proteins could function physiologically to support catalysis by bacterial NOSs.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Flavodoxina/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/biossíntese , Animais , Catálise , Cinética , Oxirredução , Especificidade da Espécie
14.
Dalton Trans ; (21): 3427-35, 2005 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-16234921

RESUMO

The NO synthases (NOSs) catalyze a two-step oxidation of L-arginine (Arg) to generate nitric oxide (NO) plus L-citrulline. Because NOSs are the only hemeproteins known to contain tetrahydrobiopterin (H4B) as a bound cofactor, the function and role of H4B in their heme-based oxygen activation and catalysis is of current interest. Distinct oxidative and reductive transitions of bound H4B cofactor occur during catalysis and are associated with distinct redox transitions of the NOS heme and flavin prosthetic groups. In this perspective, we discuss the redox transitions of H4B and heme with regard to their kinetics, regulation, role in the catalytic mechanism, and how and why they may be linked.


Assuntos
Biopterina/análogos & derivados , Heme/química , Heme/metabolismo , Óxido Nítrico Sintase/metabolismo , Animais , Fenômenos Bioquímicos , Bioquímica , Biopterina/química , Biopterina/metabolismo , Catálise , Radicais Livres/química , Radicais Livres/metabolismo , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Oxirredução
15.
Biochemistry ; 44(12): 4676-90, 2005 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-15779894

RESUMO

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.


Assuntos
Biopterina/análogos & derivados , Biopterina/química , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/metabolismo , Triptofano/química , Animais , Arginina/genética , Biopterina/metabolismo , Catálise , Dimerização , Transporte de Elétrons/genética , Estabilidade Enzimática/genética , Compostos Ferrosos/metabolismo , Heme/química , Hidroxilação , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Camundongos , Mutagênese Sítio-Dirigida , NADP/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Oxirredução , Ligação Proteica/genética , Ratos , Espectrofotometria , Triptofano/genética
16.
J Biol Chem ; 280(10): 8929-35, 2005 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-15632185

RESUMO

The nitric-oxide synthases (NOSs) make nitric oxide and citrulline from l-arginine. How the bound cofactor (6R)-tetrahydrobiopterin (H4B) participates in Arg hydroxylation is a topic of interest. We demonstrated previously that H4B radical formation in the inducible NOS oxygenase domain (iNOSoxy) is kinetically coupled to the disappearance of a heme-dioxy intermediate and to Arg hydroxylation. Here we report single turnover studies that determine and compare the kinetics of these transitions in Arg hydroxylation reactions catalyzed by the oxygenase domains of endothelial and neuronal NOSs (eNOSoxy and nNOSoxy). There was a buildup of a heme-dioxy intermediate in eNOSoxy and nNOSoxy followed by a monophasic transition to ferric enzyme during the reaction. The rate of heme-dioxy decay matched the rates of H4B radical formation and Arg hydroxylation in both enzymes. The rates of H4B radical formation differed such that nNOSoxy (18 s(-1)) > iNOSoxy (11 s(-1)) > eNOSoxy (6 s(-1)), whereas the lifetimes of the resulting H4B radical followed an opposite rank order. 5MeH4B supported a three-fold faster radical formation and greater radical stability relative to H4B in both eNOSoxy and nNOSoxy. Our results indicate the following: (i) the three NOSs share a common mechanism, whereby H4B transfers an electron to the heme-dioxy intermediate. This step enables Arg hydroxylation and is rate-limiting for all subsequent steps in the hydroxylation reaction. (ii) A direct correlation exists between pterin radical stability and the speed of its formation in the three NOSs. (iii) Uncoupled NO synthesis often seen for eNOS at low H4B concentrations may be caused by the slow formation and poor stability of its H4B radical.


Assuntos
Arginina/metabolismo , Biopterina/análogos & derivados , Biopterina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico Sintase/metabolismo , Animais , Radicais Livres/metabolismo , Heme/metabolismo , Humanos , Hidroxilação , Cinética , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Oxirredução , Ratos
18.
J Biol Chem ; 279(18): 19018-25, 2004 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-14976216

RESUMO

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.


Assuntos
Catálise , Sequência Conservada , Heme/química , Óxido Nítrico Sintase/química , Substituição de Aminoácidos , Animais , Bacillus subtilis/enzimologia , Proteínas de Bactérias , Sítios de Ligação , Ferro/química , Isoleucina , Cinética , Camundongos , Modelos Químicos , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Valina
19.
Biochem Soc Symp ; (71): 39-49, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15777011

RESUMO

NOSs (nitric oxide synthases) are flavohaem enzymes that function broadly in human health and disease. We are combining mutagenesis, crystallographic and rapid kinetic methods to understand their mechanism and regulation. The NOSs create a transient tetrahydrobiopterin radical within the enzyme to generate their free radical product (NO). Recent work is revealing how critically important this process is at all levels of catalysis. This article will synthesize four seemingly disparate but related aspects of NOS tetrahydrobiopterin radical formation: (i) how it enables productive O2 activation by providing an electron to the enzyme haem, (ii) what structural features help to regulate this electron transfer, (iii) how it enables NOS to synthesize NO from its diamagnetic substrate and (iv) how it allows NOS to release NO after each catalytic cycle instead of other nitorgen oxide-containing products.


Assuntos
Biopterina/análogos & derivados , Radicais Livres/metabolismo , Modelos Químicos , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/metabolismo , Animais , Biopterina/química , Biopterina/metabolismo , Catálise , Ativação Enzimática/fisiologia , Humanos , Óxido Nítrico/metabolismo , Oxirredução , Oxigênio/metabolismo
20.
J Biol Chem ; 278(47): 46668-73, 2003 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-14504282

RESUMO

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.


Assuntos
Arginina/análogos & derivados , Arginina/metabolismo , Biopterina/análogos & derivados , Biopterina/metabolismo , Radicais Livres/metabolismo , Óxido Nítrico Sintase/metabolismo , Animais , Arginina/química , Biopterina/química , Catálise , Clonagem Molecular , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/química , Congelamento , Cinética , Camundongos , Óxido Nítrico Sintase/química , Oxirredução , Soluções
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