Ca binding shifts dimeric dual oxidase's truncated EF-hand domain to monomer.

Hydrogen peroxide, produced by Dual Oxidase (Duox), is essential for thyroid hormone synthesis. Duox activation involves Ca binding to its EF-hand Domain (EFD), which contains two EF-hands (EFs). In this study, we characterized a truncated EFD using spectrometry, calorimetry, electrophoretic mobility, and gel filtration to obtain its Ca binding thermodynamic and kinetics, as well as to assess the associated conformational changes.

Calmodulin binding to the dehydrogenase domain of NADPH oxidase 5 alters its oligomeric state.

Superoxide generated by NADPH Oxidase 5 (Nox5) is regulated by Ca through the interaction of its self-contained Ca binding domain and dehydrogenase domain (DH). Recently, calmodulin (CaM) has been reported to enhance the Ca sensitivity of Nox5 by binding to the CaM-binding domain sequence (CMBD), in which the interaction between CaM and Nox5 is largely unclear. Here, we used the CMBD peptide and truncated DH constructs, and separately studied their interaction with CaM by fluorescence, calorimetry, and dynamic light scattering.

Binding of Nox5's EF-Hand domain to the peptides corresponding to the phosphorylatable region and regulatory inhibitory loop in its dehydrogenase domain.

Reactive oxygen species (ROS) produced by NADPH oxidase 5 (Nox5) are regulated by Ca 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.

Metal binding and conformational studies of the calcium binding domain of NADPH oxidase 5 reveal its similarity and difference to calmodulin.

The superoxide-generating activity of Nox5 is regulated by Ca flux, primarily through its self-contained calcium binding domain (EFD). Upon Ca binding, Nox5's EFD undergoes a conformational change that exposes its buried hydrophobic residues. Previously, we determined the Ca binding constants of the N-terminal half domain (N-EFD).

Polymer conjugation optimizes EDTA as a calcium-chelating agent that exclusively removes extrafibrillar minerals from mineralized collagen.

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.

Extrafibrillar collagen demineralization-based chelate-and-rinse technique bridges the gap between wet and dry dentin bonding.

Unlabelled: 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.

Engineering nitric oxide synthase chimeras to function as NO dioxygenases.

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.

The exchanged EF-hands in calmodulin and troponin C chimeras impair the Ca²⁺-induced hydrophobicity and alter the interaction with Orai1: a spectroscopic, thermodynamic and kinetic study.

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.

Characterization of the 1st and 2nd EF-hands of NADPH oxidase 5 by fluorescence, isothermal titration calorimetry, and circular dichroism.

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.

Arg375 tunes tetrahydrobiopterin functions and modulates catalysis by inducible nitric oxide synthase.

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.

Conformational States and kinetics of the calcium binding domain of NADPH oxidase 5.

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.

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

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

Lys842 in neuronal nitric-oxide synthase enables the autoinhibitory insert to antagonize calmodulin binding, increase FMN shielding, and suppress interflavin electron transfer.

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.

Catalytic reduction of a tetrahydrobiopterin radical within nitric-oxide synthase.

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.

Bacterial flavodoxins support nitric oxide production by Bacillus subtilis nitric-oxide synthase.

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).

Exploring the redox reactions between heme and tetrahydrobiopterin in the nitric oxide synthases.

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.

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.

Radical reactions of nitric oxide synthases.

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).

The three nitric-oxide synthases differ in their kinetics of tetrahydrobiopterin radical formation, heme-dioxy reduction, and arginine hydroxylation.

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.

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

Nitric-oxide synthases are flavoheme enzymes that catalyze two sequential monooxygenase reactions to generate nitric oxide (NO) from l-arginine. We investigated a possible redox role for the enzyme-bound cofactor 6R-tetrahydrobiopterin (H4B) in the second reaction of NO synthesis, which is conversion of N-hydroxy-l-arginine (NOHA) to NO plus citrulline. We used stopped-flow spectroscopy and rapid-freeze EPR spectroscopy to follow heme and biopterin transformations during single-turnover NOHA oxidation reactions catalyzed by the oxygenase domain of inducible nitric-oxide synthase (iNOSoxy).

Structure of tetrahydrobiopterin tunes its electron transfer to the heme-dioxy intermediate in nitric oxide synthase.

How 6R-tetrahydrobiopterin (H(4)B) participates in Arg hydroxylation as catalyzed by the nitric oxide synthases (NOSs) is a topic of current interest. Previous work with the oxygenase domain of inducible NOS (iNOSoxy) demonstrated that H(4)B radical formation is kinetically coupled to disappearance of an initial heme-dioxy intermediate and to Arg hydroxylation in a single turnover reaction run at 10 degrees C [Wei, C.-C.