Flavoprotein Domain Research Articles

Human MICAL1: Activation by the small GTPase Rab8 and small-angle X-ray scattering studies on the oligomerization state of MICAL1 and its complex with Rab8.

Human MICAL1 is a member of a recently discovered family of multidomain proteins that couple a FAD-containing monooxygenase-like domain to typical protein interaction domains. Growing evidence implicates the NADPH oxidase reaction catalyzed by the flavoprotein domain in generation of hydrogen peroxide as a second messenger in an increasing number of cell types and as a specific modulator of actin filaments stability. Several proteins of the Rab families of small GTPases are emerging as regulators of MICAL activity by binding to its C-terminal helical domain presumably shifting the equilibrium from the free - auto-inhibited - conformation to the active one. We here extend the characterization of the MICAL1-Rab8 interaction and show that indeed Rab8, in the active GTP-bound state, stabilizes the active MICAL1 conformation causing a specific four-fold increase of kcat of the NADPH oxidase reaction. Kinetic data and small-angle X-ray scattering (SAXS) measurements support the formation of a 1:1 complex between full-length MICAL1 and Rab8 with an apparent dissociation constant of approximately 8 μM. This finding supports the hypothesis that Rab8 is a physiological regulator of MICAL1 activity and shows how the protein region preceding the C-terminal Rab-binding domain may mask one of the Rab-binding sites detected with the isolated C-terminal fragment. SAXS-based modeling allowed us to propose the first model of the free full-length MICAL1, which is consistent with an auto-inhibited conformation in which the C-terminal region prevents catalysis by interfering with the conformational changes that are predicted to occur during the catalytic cycle.

Restricting the conformational freedom of the neuronal nitric-oxide synthase flavoprotein domain reveals impact on electron transfer and catalysis

The signaling molecule nitric oxide (NO) is synthesized in animals by structurally related NO synthases (NOSs), which contain NADPH/FAD- and FMN-binding domains. During catalysis, NADPH-derived electrons transfer into FAD and then distribute into the FMN domain for further transfer to internal or external heme groups. Conformational freedom of the FMN domain is thought to be essential for the electron transfer (ET) reactions in NOSs. To directly examine this concept, we utilized a "Cys-lite" neuronal NOS flavoprotein domain and substituted Cys for two residues (Glu-816 and Arg-1229) forming a salt bridge between the NADPH/FAD and FMN domains in the conformationally closed structure to allow cross-domain disulfide bond formation or cross-linking by bismaleimides of various lengths. The disulfide bond cross-link caused a ≥95% loss of cytochrome c reductase activity that was reversible with DTT treatment, whereas graded cross-link lengthening gradually increased activity, thus defining the conformational constraints in the catalytic process. We used spectroscopic and stopped-flow techniques to further investigate how the changes in FMN domain conformational freedom impact the following: (i) the NADPH interaction; (ii) kinetics of electron loading (flavin reduction); (iii) stabilization of open versus closed conformational forms in two different flavin redox states; (iv) reactivity of the reduced FMN domain toward cytochrome c; (v) response to calmodulin binding; and (vi) the rates of interflavin ET and the FMN domain conformational dynamics. Together, our findings help explain how the spatial and temporal behaviors of the FMN domain impact catalysis by the NOS flavoprotein domain and how these behaviors are governed to enable electron flow through the enzyme.

Subunit NDUFV3 is present in two distinct isoforms in mammalian complex I

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the electron transport chain in mammalian mitochondria. Extensive proteomic and structural analyses of complex I from Bos taurus heart mitochondria have shown it comprises 45 subunits encoded on both the nuclear and mitochondrial genomes; 44 of them are different and one is present in two copies. The bovine heart enzyme has provided a model for studying the composition of complex I in other mammalian species, including humans, but the possibility of additional subunits or isoforms in other species or tissues has not been explored. Here, we describe characterization of the complexes I purified from five rat tissues and from a rat hepatoma cell line. We identify a~50kDa isoform of subunit NDUFV3, for which the canonical isoform is only ~10kDa in size. We combine LC-MS and MALDI-TOF mass spectrometry data from two different purification methods (chromatography and immuno-purification) with information from blue native PAGE analyses to show the long isoform is present in the mature complex, but at substoichiometric levels. It is also present in complex I in cultured human cells. We describe evidence that the long isoform is more abundant in both the mitochondria and purified complexes from brain (relative to in heart, liver, kidney and skeletal muscle) and more abundant still in complex I in cultured cells. We propose that the long 50kDa isoform competes with its canonical 10kDa counterpart for a common binding site on the flavoprotein domain of complex I.

Tetrahydrobiopterin redox cycling in nitric oxide synthase: evidence supports a through-heme electron delivery.

The nitric oxide synthases (NOS) catalyze a two-step oxidation of l-arginine (Arg) to generate NO. In the first step, O2 activation involves one electron being provided to the heme by an enzyme-bound 6R-tetrahydro-l-biopterin cofactor (H4 B), and the H4 B radical must be reduced back to H4 B in order for NOS to continue catalysis. Although an NADPH-derived electron is used to reduce the H4 B radical, how this occurs is unknown. We hypothesized that the NOS flavoprotein domain might reduce the H4 B radical by utilizing the NOS heme porphyrin as a conduit to deliver the electron. This model predicts that factors influencing NOS heme reduction should also influence the extent and rate of H4 B radical reduction in kind. To test this, we utilized single catalytic turnover and stop-freeze methods, along with electron paramagnetic resonance spectroscopy, to measure the rate and extent of reduction of the 5-methyl-H4 B radical formed in neuronal NOS (nNOS) during Arg hydroxylation. We used several nNOS variants that supported either a slower or faster than normal rate of ferric heme reduction. We found that the rates and extents of nNOS heme reduction correlated well with the rates and extents of 5-methyl-H4 B radical reduction among the various nNOS enzymes. This supports a model where the heme porphyrin transfers an electron from the NOS flavoprotein to the H4 B radical formed during catalysis, revealing that the heme plays a dual role in catalyzing O2 activation or electron transfer at distinct points in the reaction cycle.

Properties and catalytic activities of MICAL1, the flavoenzyme involved in cytoskeleton dynamics, and modulation by its CH, LIM and C-terminal domains

MICAL1 is a cytoplasmic 119 kDa protein participating in cytoskeleton dynamics through the NADPH-dependent oxidase and F-actin depolymerizing activities of its N-terminal flavoprotein domain, which is followed by calponin homology (CH), LIM domains and a C-terminal region with Pro-, Glu-rich and coiled-coil motifs. MICAL1 and truncated forms lacking the C-terminal, LIM and/or CH regions have been produced and characterized. The CH, LIM and C-terminal regions cause an increase of Km,NADPH exhibited by the NADPH oxidase activity of the flavoprotein domain, paralleling changes in the overall protein charge. The C-terminus also determines a ∼10-fold decrease of kcat, revealing its role in establishing an inactive/active conformational equilibrium, which is at the heart of the regulation of MICAL1 in cells. F-actin lowers Km,NADPH (10–50 μM) and increases kcat (10–25 s−1) to similar values for all MICAL forms. The apparent Km,actin of MICAL1 is ∼10-fold higher than that of the other forms (3–5 μM), reflecting the fact that F-actin binds to the flavoprotein domain in the MICAL's active conformation and stabilizes it. Analyses of the reaction in the presence of F-actin indicate that actin depolymerization is mediated by H2O2 produced by the NADPH oxidase reaction, rather than due to direct hydroxylation of actin methionine residues.

Oral 1832-2 - Charge pairing and phosphorylation regulate the conformational equilibrium and switching rates in neuronal and endothelial NO synthase flavoprotein domains

Oral 1832-2 - Charge pairing and phosphorylation regulate the conformational equilibrium and switching rates in neuronal and endothelial NO synthase flavoprotein domains

Charge-pairing interactions control the conformational setpoint and motions of the FMN domain in neuronal nitric oxide synthase

The NOS (nitric oxide synthase; EC 1.14.13.39) enzymes contain a C-terminal flavoprotein domain [NOSred (reductase domain of NOS)] that binds FAD and FMN, and an N-terminal oxygenase domain that binds haem. Evidence suggests that the FMN-binding domain undergoes large conformational motions to shuttle electrons between the NADPH/FAD-binding domain [FNR (ferredoxin NADP-reductase)] and the oxygenase domain. Previously we have shown that three residues on the FMN domain (Glu762, Glu816 and Glu819) that make charge-pairing interactions with the FNR help to slow electron flux through nNOSred (neuronal NOSred). In the present study, we show that charge neutralization or reversal at each of these residues alters the setpoint [Keq(A)] of the NOSred conformational equilibrium to favour the open (FMN-deshielded) conformational state. Moreover, computer simulations of the kinetic traces of cytochrome c reduction by the mutants suggest that they have higher conformational transition rates (1.5-4-fold) and rates of interflavin electron transfer (1.5-2-fold) relative to wild-type nNOSred. We conclude that the three charge-pairing residues on the FMN domain govern electron flux through nNOSred by stabilizing its closed (FMN-shielded) conformational state and by retarding the rate of conformational switching between its open and closed conformations.

Aluminum‐substituted heme domain of P450BM‐3 (BMP): Introducing a heme‐derived fluorescent probe for studies of substrate binding and protein–protein interactions in cytochromes P450

We describe a novel technique for heme removal and replacement in the heme domain of P450BM-3 (BMP). The method was applied to obtain the aluminum-protoporphyrin IX (Al-PP) substituted derivative of BMP (Al-BMP). The overall yield of the purified Al-BMP was about 15% as related to the initial amount of the hemeprotein. Al-BMP possesses extensive fluorescence in the 550-650 nm region with excitation in the porphyrin absorbance bands. The protein was shown to bind substrates of P450BM-3 (palmitic, arachidonic, and cis-parinaric acids) with affinities similar to those of the native enzyme (3-6 µM). However, the substrate-induced changes in fluorescence of Al-PP reveal the existence of a second, low-affinity substrate-binding site, which cannot be detected by the spin shift in the native, heme-containing BMP. Using fluorescence resonance energy transfer, we have demonstrated that Al-BMP forms a complex with the flavoprotein domain of P450BM-3 labeled with 7-ethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin maleimide, revealing the affinity similar to that of native BMP (Kd = 5 µM at 0.06 M ionic strength). Therefore, aluminum-substituted BMP may serve as a valuable tool in studies on the mechanisms of interactions of P450s with their substrates and protein partners.

Express Your LOV: An Engineered Flavoprotein as a Reporter for Protein Expression and Purification

In this work, we describe the utility of Light, Oxygen, or Voltage-sensing (LOV) flavoprotein domains from plant phototropins as a reporter for protein expression and function. Specifically, we used iLOV, an enhanced and more photostable variant of LOV. A pET-based plasmid for protein expression was constructed, encoding a C terminal iLOV-octahistidine (His8)-tag and a HRV 3C protease cleavage recognition site. Ten different proteins, with various sub-cellular locations, were cloned into the plasmid, creating iLOV-His8 tag fusions. To test protein expression and how iLOV could be used as a reporter, the proteins were expressed in three different cell lines, in four different culture media, at two different temperatures. To establish whether the presence of the iLOV tag could have an impact on the functionality, one of the proteins, EspG, was over-expressed and purified. EspG is an “effector” protein normally produced by enterohemorrhagic E. coli strains and “injected” into host cells via the T3SS. We tested functionality of EspG-iLOV fusion by performing functional studies of EspG in mammalian host cells. When EspG-iLOV was microinjected into the host cell, the Golgi apparatus was completely disrupted as had previously been observed for EspG.

Calcium‐Dependent Interactions and Electron Transfer in NADPH Oxidase 5

Superoxide generated by NADPH oxidase 5 (NOX 5) is of growing importance for physiology and pathobiology. The NOX5 activity appears to be regulated by its self‐contained calcium binding domain (CaBD), through an interaction with its flavoprotein domain. Here, using fluorescence we showed that such domain‐domain interaction is Ca2+‐dependent. The resulting interaction enhances the electron transfer from NADPH to FAD in cytochrome C assay and gates heme reduction using full‐length NOX5. Our data support the published results showing that CaBD acts as an activator by binding the inhibitory loops in the flavoprotein, and thus lifting the blockage of electron transfer for superoxide production. CaBD contains two half domains, in which the N‐terminal half contains the Ca2+‐specific EF‐hands and The C‐terminal half contains the Ca2+/Mg2+ mixed EF‐hands. The half domains alone showed no interaction with the flavoprotein. However, the calcium knock‐out mutant, CaBD(E99Q/E143Q) in which the E residues at the –z position in 3rd and 4th EF‐hands were replaced with Q, retains its interaction with the flavoprotein, suggesting that its N‐terminal half is critical for regulating electron transfer. Currently we are investigating other constructs, such as CaBD(E31Q/E64Q) and transmembrane‐deleted NOX5, for the studies of domain‐domain interaction and electron transfer. Furthermore, we include calmodulin (CaM) in our existing system to investigate a possible ternary complex formation (CaBD/flavoprotein/CaM) using FRET and for determining its effects on electron transfer as it has been shown that CaM interacts with the flavoprotein. This work is supported in part by National Science Foundation CCLI grant.

Control Of Electron Transfer And Catalysis In Neuronal NOS By A Hinge Connecting The FMN And FNR Domains

In nitric oxide synthase enzymes (NOSs) two flexible hinge elements connect the FMN domain to the rest of the enzyme and may guide FMN domain interactions with its partner FNR and NOSoxy domains to enable the electron transfers required during catalysis. In our previous study, we found that incorporating the nNOS FMN‐FNR hinge into eNOS increased its NO synthesis activity 4‐fold, decreased uncoupled NADPH oxidation, and supported faster heme reduction (kr), implying that the composition and/or length of the FMN‐FNR hinge is important for determining electron transfer and catalysis. To more systematically investigate the role of the FMN‐FNR hinge we constructed mutants in nNOS that either shorten or lengthen the hinge by 2, 4, and 6 residues. We found that FMN‐FNR hinge length helps determine electron flux through the nNOS flavoprotein domain. Shortening the hinge inhibited electron flux through both the calmodulin (CaM)‐free and CaM‐bound enzymes, while lengthening the hinge relieved the repression on electron flux that is normally present in the CaM‐free enzyme, and has either no impact or was deleterious for electron flux through the CaM‐bound enzyme. The nNOS kr was also sensitive to FMN‐FNR hinge length. Small changes in hinge length were tolerated, while changes beyond 2 or 4 residues were deleterious to kr, and also decoupled nNOS NADPH consumption from NO synthesis. Supported by NIH GM51941 & HL63744.

A kinetic model linking protein conformational motions, interflavin electron transfer and electron flux through a dual‐flavin enzyme – simulating the reductase activity of the endothelial and neuronal nitric oxide synthase flavoprotein domains

NADPH-dependent dual-flavin enzymes provide electrons in many redox reactions, although the mechanism responsible for regulating their electron flux remains unclear. We recently proposed a four-state kinetic model that links the electron flux through a dual-flavin enzyme to its rates of interflavin electron transfer and FMN domain conformational motion [Stuehr DJ et al. (2009) FEBS J276, 3959-3974]. In the present study, we ran computer simulations of the kinetic model to determine whether it could fit the experimentally-determined, pre-steady-state and steady-state traces of electron flux through the neuronal and endothelial NO synthase flavoproteins (reductase domains of neuronal nitric oxide synthase and endothelial nitric oxide synthase, respectively) to cytochrome c. We found that the kinetic model accurately fitted the experimental data. The simulations gave estimates for the ensemble rates of interflavin electron transfer and FMN domain conformational motion in the reductase domains of neuronal nitric oxide synthase and endothelial nitric oxide synthase, provided the minimum rate boundary values, and predicted the concentrations of the four enzyme species that cycle during catalysis. The findings of the present study suggest that the rates of interflavin electron transfer and FMN domain conformational motion are counterbalanced such that both processes may limit electron flux through the enzymes. Such counterbalancing would allow a robust electron flux at the same time as keeping the rates of interflavin electron transfer and FMN domain conformational motion set at relatively slow levels.

Electron transfer flavoprotein domain II orientation monitored using double electron‐electron resonance between an enzymatically reduced, native FAD cofactor, and spin labels

Human electron transfer flavoprotein (ETF) is a soluble mitochondrial heterodimeric flavoprotein that links fatty acid β-oxidation to the main respiratory chain. The crystal structure of human ETF bound to medium chain acyl-CoA dehydrogenase indicates that the flavin adenine dinucleotide (FAD) domain (αII) is mobile, which permits more rapid electron transfer with donors and acceptors by providing closer access to the flavin and allows ETF to accept electrons from at least 10 different flavoprotein dehydrogenases. Sequence homology is high and low-angle X-ray scattering is identical for Paracoccus denitrificans (P. denitrificans) and human ETF. To characterize the orientations of the αII domain of P. denitrificans ETF, distances between enzymatically reduced FAD and spin labels in the three structural domains were measured by double electron-electron resonance (DEER) at X- and Q-bands. An FAD to spin label distance of 2.8 ± 0.15 nm for the label in the FAD-containing αII domain (A210C) agreed with estimates from the crystal structure (3.0 nm), molecular dynamics simulations (2.7 nm), and rotamer library analysis (2.8 nm). Distances between the reduced FAD and labels in αI (A43C) were between 4.0 and 4.5 ± 0.35 nm and for βIII (A111C) the distance was 4.3 ± 0.15 nm. These values were intermediate between estimates from the crystal structure of P. denitrificans ETF and a homology model based on substrate-bound human ETF. These distances suggest that the αII domain adopts orientations in solution that are intermediate between those which are observed in the crystal structures of free ETF (closed) and ETF bound to a dehydrogenase (open).

Identification of Two Novel Reductases Involved in Equol Biosynthesis in Lactococcus Strain 20–92

Lactococcus strain 20–92 is a bacterium that produces equol directly from daidzein under anaerobic conditions. In this study, we reveal that the transcription of the gene encoding daidzein reductase in Lactococcus strain 20–92 (L-DZNR), which is responsible for the first stage of the biosynthesis of equol from daidzein, is regulated by the presence of daidzein. We analyzed the sequence surrounding the L-DZNR gene and found six novel genes, termed orf-US4, orf-US3, orf-US2, orf-US1, orf-DS1 and orf-DS2. These genes were expressed in Escherichia coli, and the resulting gene products were assayed for dihydrodaidzein reductase (DHDR) and tetrahydrodaidzein reductase (THDR) activity. The results showed that orf-US2 and orf-US3 encoded DHDR and THDR, respectively. DHDR in Lactococcus strain 20–92 (L-DHDR) was similar to the 3-oxoacyl-acyl-carrier-protein reductases of several bacteria and belonged to the short chain dehydrogenase/reductase family. THDR in Lactococcus strain 20–92 (L-THDR) was similar to several putative fumarate reductase/succinate dehydrogenase flavoprotein domain proteins. L-DHDR required NAD(P)H for its activity, whereas L-THDR required neither NADPH nor NADH. Thus, we succeeded in identifying two novel enzymes that are related to the second and third stages of the biosynthetic pathway that converts daidzein to equol.

Characterization of an NADH-Dependent Persulfide Reductase from Shewanella loihica PV-4: Implications for the Mechanism of Sulfur Respiration via FAD-Dependent Enzymes,

The NADH-dependent persulfide reductase (Npsr), a recently discovered member of the PNDOR family of flavoproteins that contains both the canonical flavoprotein reductase domain and a rhodanese domain, is proposed to be involved in the dissimilatory reduction of S(0) for Shewanella loihica PV-4. We have previously shown that polysulfide is a substrate for this enzyme, and a recently determined structure of a closely related enzyme (CoADR-Rhod from Bacillus anthracis) suggested the importance of a bound coenzyme A in the mechanism. The work described here shows that the in vivo oxidizing substrates of Npsr are the persulfides of small thiols such as CoA and glutathione. C43S, C531S, and C43,531S mutants were created to determine the role of the flavoprotein domain cysteine (C43) and the rhodanese domain cysteine (C531) in the mechanism. The absolute requirement for C43 in persulfide or DTNB reductase activity shows that this residue is involved in S-S bond breakage. C531 contributes to, but is not required for, catalysis of DTNB reduction, while it is absolutely required for reduction of any persulfide substrates. Titrations of the enzyme with NADH, dithionite, titanium(III), or TCEP demonstrate the presence of a mixed-disulfide between C43 and a tightly bound CoA, and structures of the C43 and C43,531S mutants confirm that this coenzyme A remains tightly bound to the enzyme in the absence of a C43-CoA S-S bond. The structure of Npsr suggests a likely site for binding and reaction with the persulfide substrate on the rhodanese domain. On the basis of kinetic, titration, and structural data, a mechanism for the reduction of persulfides by Npsr is proposed.

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. 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.

CDNA Cloning, Sequence Identification and Tissue Expression Distribution of Three Novel Genes: DFP, ITM2B and PQLC1 from Black-Boned Sheep (Ovis Aries)

ABSTRACTThe complete coding sequences of three of Black-boned sheep (Ovis aries) genes DFP, ITM2B and PQLC1 were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR) according to the conserved sequence information of the cattle or other mammals and known highly homologous sheep ESTs. Black-boned sheep DFP gene encodes a protein of 213 amino acids which contains the conserved putative DNA/pantothenate metabolism flavoprotein domain and has high homology with the DFP proteins of four species: cattle (99%), dog (91%), human (88%) and zebrafish (53%). Black-boned sheep ITM2B gene encodes a protein of 266 amino acids that contains the conserved putative BRICHOS domain and has high similarity with the ITM2B proteins of five species: cattle (98%), human (95%), mouse (94%), rat (94%) and pig (94%). Black-boned sheep PQLC1 gene encodes a protein of 253 amino acids that contains the conserved putative PQ-loop repeat domain and has high homology with the PQLC1 proteins of four species: cattle (99%), human (91%), mouse (90%) and rat (84%). The phylogenetic tree analysis demonstrated that Black-boned sheep's DFP, ITM2B andPQLC1 proteins have a close relationship with cattle. The tissue expression analysis indicated that Black-boned sheep DFP, ITM2B and PQLC1 genes were expressed in a range of tissues including leg muscle, kidney, skin, longissimus dorsi muscle, spleen, heart and liver. Our experiment is the first to provide the primary foundation for further insight into these three sheep genes.

Structural and mechanistic aspects of flavoproteins: electron transfer through the nitric oxide synthase flavoprotein domain

Nitric oxide synthases belong to a family of dual-flavin enzymes that transfer electrons from NAD(P)H to a variety of heme protein acceptors. During catalysis, their FMN subdomain plays a central role by acting as both an electron acceptor (receiving electrons from FAD) and an electron donor, and is thought to undergo large conformational movements and engage in two distinct protein-protein interactions in the process. This minireview summarizes what we know about the many factors regulating nitric oxide synthase flavoprotein domain function, primarily from the viewpoint of how they impact electron input/output and conformational behaviors of the FMN subdomain.

Characterization of the Calcium Binding Domain of NADPH Oxidase 5 (NOX5)

Superoxide generated by non-phagocyte NADPH oxidases (NOXs), such as NOX5, is of growing importance for vascular physiology and pathology. NOX5 enzyme consists of a transmembrane heme domain that is linked to a flavoprotein domain that contains FAD and binds NADPH. It appears to be regulated by self-contained Ca2+ binding domains (CaBD), which contains four EF-hands motifs. Previously we demonstrated that this calcium binding gates the heme reduction in NOX5, possibly thorough the CaBD-flavorprotein interaction. To better understand its structure and function, here we characterized the metal binding properties of the recombinant CaBD by fluorescent spectroscopy. Our data revealed that CaBD binds to Ca2+, Mg2+, terbium (Tb3+) in the range of μM to mM. The data are further supported by the studies using the site-directed labeled CaBD. The rate of calcium association was too fast to be determined by a stopped-flow device, but the dissociation rate constant was determined to be 5 s−1 at 20 °C. The ANS titration and Stern-Volmer plots suggested that there was a significant conformational change upon the metal bindings. Interestingly, the spectra of circular dichorism indicated otherwise no change on the context of its secondary structure. However, this conformational change can be observed using the Surface Plasmon Resonance with the CaBD immobilized in the sensor chip. Because our data and other studies suggest there are two different types of calcium bindings in CaBD, currently we are performing the similar studies using N- and C-terminal halves of CaBD (aa 1-78 and 79-184), and mutants. We also are investigating the metal bindings in the CaBD of Dual Oxidase (DUOX), in which its hydrogen peroxide activity is controlled by calcium binding.

Regulation of the phagocyte NADPH oxidase activity: phosphorylation of gp91phox/NOX2 by protein kinase C enhances its diaphorase activity and binding to Rac2, p67phox, and p47phox

Neutrophils generate microbicidal oxidants through activation of a multicomponent enzyme called NADPH oxidase. During activation, the cytosolic NADPH oxidase components (p47(phox), p67(phox), p40(phox), and Rac2) translocate to the membranes, where they associate with flavocytochrome b(558), which is composed of gp91(phox)/NOX2 and p22(phox), to form the active system. During neutrophil stimulation, p47(phox), p67(phox), p40(phox), and p22(phox) are phosphorylated; however, the phosphorylation of gp91(phox)/NOX2 and its potential role have not been defined. In this study, we show that gp91(phox) is phosphorylated in stimulated neutrophils. The gp91(phox) phosphoprotein is absent in neutrophils from chronic granulomatous disease patients deficient in gp91(phox), which confirms that this phosphoprotein is gp91(phox). The protein kinase C inhibitor GF109203X inhibited phorbol 12-myristate 13-acetate-induced phosphorylation of gp91(phox), and protein kinase C (PKC) phosphorylated the recombinant gp91(phox)- cytosolic carboxy-terminal flavoprotein domain. Two-dimensional tryptic peptide mapping analysis showed that PKC phosphorylated the gp91(phox)-cytosolic tail on the same peptides that were phosphorylated on gp91(phox) in intact cells. In addition, PKC phosphorylation increased diaphorase activity of the gp91(phox) flavoprotein cytosolic domain and its binding to Rac2, p67(phox), and p47(phox). These results demonstrate that gp91(phox) is phosphorylated in human neutrophils by PKC to enhance its catalytic activity and assembly of the complex. Phosphorylation of gp91(phox)/NOX2 is a novel mechanism of NADPH oxidase regulation.