nadph oxidase structure

The very first such prokaryotic enzyme is NADPH oxidase, which converts molecular oxygen to superoxide. 2007 Dec 1; 43(11): 1469–1483. The phagocyte NADPH oxidase is a multicomponent enzyme consisting of membrane-bound as well as cytosolic subunits. This conformational change might reflect the absence of the FAD-binding domain in the human NOX2 partial structure. Spectral changes were recorded with a stopped-flow instrument after mixing anaerobically reduced csTM (1.3–2.4 μM enzyme/0.5 mM dithionite) with dioxygen. transfer of an electron from cytosolic NADPH to oxygen to generate ROS at the extracellular face . (E) Superposition of csDH with ferredoxin-NADP oxidoreductase enzymes. Rather, the regulatory role of strictly conserved Phe693 is predicted to emerge only in the context of the full-length protein. 220 R. Ray and A. M. Shah Figure 2 Schematic diagram of the structure of the active NADPH oxidase complex The classical NADPH oxidase comprises a membrane-bound gp91phox/p22phox heterodimer and other subunits (p67phox,p47phox,p40phox and Rac) which associate with this complex in the activated enzyme. The functional importance of Arg256 and His317 is further documented by disease-inducing mutations affecting the corresponding residues of human NOX2. We thank the Swiss Light Source, European Synchrotron Radiation Facility, and Deutsches Elektronen-Synchrotron for providing synchrotron radiation facilities, and their staff for supervising data collection; Stefano Rovida and Federico Forneris for providing technical support with inhibition assays and crystallographic analyses; Thomas Schneider (European Molecular Biology Laboratory–Deutsches Elektronen-Synchrotron, Hamburg) for his help and assistance; and Claudia Binda and Federico Forneris for critical reading of the manuscript. (C) NADPH-oxidase activity of the isolated csDH. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1702293114/-/DCSupplemental. Branisteanu DC, Branisteanu DE, Feraru CI, Branisteanu CI, Moraru A, Zemba M, Balta F. Exp Ther Med. S1A) (14, 15). (B) Overall and (C) closed-up views of the interacting surfaces between DH (light orange) and TM (light blue) of csNOX5 (Fig. The initial rate of enzyme activity was calculated over the first 30–120 s of exposure to NADPH. Figure 1. The small G protein carries an essential role in the activation of the oxidase by switching between a GDP-bound (inactive) and GTP-linked (activ… 5 A and B). It is a NAD(P)H and a NADP.It is a conjugate acid of a NADPH(4-). The substitution Phe693Ser showed a twofold increase in Vmax compared with the WT, whereas the deletion of Phe693 did not elicit any remarkable change on the steady-state kinetic properties of the DH domain (Table S2). Reaction with dioxygen of csTM domain. In this way, one heme lies proximal to the cytosolic (inner) side of the domain, whereas the second heme is located toward the outer extracytoplasmic side. The Tm of csDH was measured by thermal shift experiments. The activation of phagocytic NADPH oxidase occurs through a complex series of protein interactions. NOXs are membrane proteins that share the same catalytic core: a six transmembrane helical domain (TM) and a C-terminal cytosolic dehydrogenase domain (DH). The RCSB PDB also provides a variety of tools and resources. Given the high conservation of the C-terminal residues, similar mechanisms to convey regulatory signals to the catalytic core might be operational also in other NOXs (33, 34) (Fig. phox. 48 . NADPH oxidase is another major site of ROS production and its contribution to DM-in-duced ROS increase has been elucidated not only in vascular smooth muscle cells and endothelial cells, but also in cardiomyocytes. Moreover, this first manually built model positioned the B and D loops of TM in direct contact with the loops surrounding the flavin- and NADP-binding sites of DH. Upon increase of intracellular Ca2+, the N-terminal EF-hand domain binds to and activates NOX5. This site needs JavaScript to work properly. Epub 2001 Dec 3. The resin was washed with lysis buffer containing 0.05% (wt/vol) DDM and eluted with elution buffer [50 mM Hepes pH 7.5, 300 mM NaCl, 5% (vol/vol) glycerol, 0.05% DDM, 150 mM imidazole]. Superoxide anion generation was determined by photoemission every 30 s for 5–10 min. Next, 1 mM sodium dithionite was mixed with 6 μM csTM in storage buffer with or without 5 mM sodium cyanide and let react. The crucial feature of this mechanistic proposal is that NADPH-oxidation at the flavin site takes place only when the enzyme is in the active conformation, thus preventing the risk of NADPH-derived electrons being diverted to nonproductive redox reactions. Predicted interactions between DH and TM. All of the spectra were recorded with Agilent Diode Array at 25 °C. Enter multiple addresses on separate lines or separate them with commas. phox, also termed as NOX2, is present with several The elucidation of NOX 3D structure outlines a general scheme for NOX regulation with the C-terminal residues functioning as regulatory toggle switch. Protein expression, purification, mutant preparation, and enzymatic assays are described in SI Materials and Methods. Research in the authors’ laboratory is supported by the Associazione Italiana per la Ricerca sul Cancro (IG-15208) and the Italian Ministry for University and Research (PRIN2015-20152TE5PK_004). Our results highlight key structural elements common to the entire NOX family, such as the toggle-switch at the C terminus and the dioxygen binding pocket. NADPH is a coenzyme that contributes to multiple biological reactions by supplying electrons. 470–500 nm/emission (Em.) 2 and Fig. The powerful production (or its deregulation/deficiency) of ROS by NOXs underlies pathological conditions, such as oxidative stress, malignancies, neurodegenerative disease, senescence, and chronic granulomatous disease (1⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓–12) (Fig. For example, in the case of NOX5, the regulatory calmodulin- and EF-hand binding segments are located in proximity of the C-terminal residues and NADPH-binding site (Fig. NADPH oxidase plays a fundamental role in maintaining normal cell functions and can be activated by diverse stimuli, such as chemical, physical, environmental, and biological factors.  |  They are instead actively generated by NADPH oxidases (NOXs), a family of highly regulated enzymes that underpin complex functions in the control of cell proliferation and antibacterial defense. Ciobotaru OR, Stoleriu G, Ciobotaru OC, Grigorovici A, Voinescu DC, Matei MN, Cobzaru RG, Manolache N, Lupu MN. Here, we describe crystal structures of DH and TM, forming the catalytic core common to the whole NOX family. The mutated residues are scattered throughout the protein. The FAD-binding lobe is in orange and the NADPH-binding lobe in gray. 5 A and B and Fig. The TM part of csNOX5 (residues 209–412; csTM) was inserted into a modified pET24b carrying an N-terminal FLAG-(His)8-SUMO tag. It can be found in the plasma membrane as well as in the membranes of phagosomes used by neutrophil white blood cells to engulf microorganisms. S5). The fluorescence was monitored over a temperature gradient from 20 to 90 °C reading every 0.5 °C [Instrument settings: FAD, excitation (Ex.) 2014;5:e27952. NADPH Oxidase: Structure and Function NOX plays a pivotal role in the production of ROS and, in particular, of the superoxide anion (O 2 − ) at the expense of NADPH. Interdomain regulatory interactions in NOX. Methods are included for testing assembly and function of multicomponent oxidase complexes and for analyzing reactive oxygen species (ROS) generation in different systems by various means, while addressing pitfalls of ROS probes currently being used, as well as protocols on NADPH oxidase regulation and their function in cells. All subsequent steps were carried out at 4 °C. The TM domain of csNOX5 (residues 209–412) was crystallized in a lipid mesophase, which provides a better crystallization environment for membrane proteins (20). (A) Schematic representation of the domain organization of NOX5. The domain structures were then docked in silico to provide a generic model for the NOX family. Once the cells reached OD600 = 1.2, protein expression was induced with 0.4 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 16 h at 17 °C. To make the stopped-flow spectrometer anaerobic, the flow-circuit of this apparatus was repeatedly washed with anaerobic buffer. NADPH oxidase in normal CNS processes, as well as a num-ber of brain diseases (85, 113), still relatively little is known about the expression, regulation, and function of this enzyme complex in cerebral cells or tissues. The eluted protein was treated with SUMO protease overnight and passed again onto TALON resin to eliminate the purification tag. 1C and Table S2). (B) Overall view of csDH with bound FAD (carbons in yellow). In this way, the flavin dimethybenzene ring and a heme propionate resulted to be in van der Waals contact, consistent with electron transfer directly occurring across the two prosthetic groups. Notably, whereas the R256S mutant displayed the same apparent melting temperature (Tm) as the WT (61 °C), the H317R variant showed lower protein stability (appTm = 43.5 °C) (Table S4). Peak fractions were pooled and concentrated to 7–10 mg/mL using the following extinction coefficients: ε280 = 48.150 M−1 cm−1 for csDH and ε280 = 48.650 M−1 cm−1 for csDH-PWLELAAA. The weighted 2Fo–Fc electron density maps are contoured at 1.4 σ levels. Haddock allows the implementation of user-defined restrains. It participates in the regulation of cellular pH and is blocked by zinc. This cavity is lined by the propionate 7 of the heme (Fisher nomenclature) and the strictly conserved residues Arg256, His317, and iron-coordinating His313. Oxygens are in red, nitrogens in blue, and phosphorous in orange. If under physiological conditions some processes are beneficial and necessary for life, under pathophysiological conditions they are noxious, harmful. Thank you for your interest in spreading the word on PNAS. Strategies for identifying synthetic peptides to act as inhibitors of NADPH oxidases, or "all that you did and did not want to know about Nox inhibitory peptides". The structure of the regulatory domain of a plant NADPH oxidase is available, OsRBOH-B, a close relative to NOX5 [ 30 ]. Online ISSN 1091-6490. To obtain a solution containing 9 μM dioxygen, the proper volumes of anaerobic and air-saturated buffers were mixed.  |  So far, seven isoforms of the NADPH oxidases have been identified: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1, and Duox2. During the resting state, the multidomain regulatory subunits p40P(phox), p47(phox), p67(Phox) are located in the cytosol organized as a complex. 2020 Sep;20(3):2368-2372. doi: 10.3892/etm.2020.8792. Here, we focus on NADPH oxidases, a family of . Postanesthetic skin erythema due to succinylcholine versus atracurium. The authors declare no conflict of interest. This observation implies that Phe693 has a limited influence on the catalysis of the isolated DH domain, which is in a deregulated active state. 4A). The essence of this mechanistic model is that the regulatory cues conformationally gate NADPH-binding, implicitly providing a handle for activating/deactivating the very first step in the redox chain. Insets show the corresponding stopped-flow traces at the characteristic absorbance maxima for oxidized or reduced enzyme (see Fig. 3 A–C). It works as a reducing agent in lipid and nucleic acid synthesis. Initial apparent velocities were plotted against NADPH concentration using Michaelis–Menten equation to calculate Km and Kcat (GraphPad Prism software). NADPH (green carbons) is modeled with the nicotinamide stacking against the isoalloxazine moiety of FAD (yellow carbons) by similarity with spinach ferredoxin-reductase (16). Small GTPases. The domain is depicted in orange with the calmodulin-binding region in blue (R644-V663) (Fig. S1B). They were processed with XDS (36) and scaled with aimless (37). Sequence and structural alignments of NOXs. The desalted protein was injected into a MonoQ column (GE Healthcare) equilibrated in the same buffer. Users can perform simple and advanced searches based on annotations relating to sequence, structure and function. Nicotinamide adenine dinucleotide phosphate is a form of NADP+ . The activity of the purified csDH domain was measured under aerobic condition following the oxidation of NADPH at 340 nm (ε = 6.22 M−1 cm−1) in a Cary 100 UV-visible spectrophotometer (Varian) equipped with a thermostated cell holder (T = 25 °C). In particular, the C terminus functions as a toggle switch, which affects access of the NADPH substrate to the enzyme. S2). Specifically, reduced enzymes were prepared by injecting 200 mM sodium dithionite (5 μL/mL enzyme) into the anaerobic vial using a gas-tight Hamilton syringe. Moreover, Arg360 and Lys361 on loop D are modeled in direct contact with the C-terminal Phe693 of DH, in the core of the nicotinamide-binding site (Fig. Two transmembrane subunits, gp91 phox and p22 phox, make up flavocytochrome b 558, the catalytic core of the complex.This heterodimer catalyzes the transfer of electrons from cytosolic NADPH to molecular oxygen, thus generating … In all cases, the assay was performed in 20-μL final volume using 15 μM protein in LSbuffer. Branisteanu DC, Stoleriu G, Branisteanu DE, Boda D, Branisteanu CI, Maranduca MA, Moraru A, Stanca HT, Zemba M, Balta F. Exp Ther Med. ROS are small oxygen-derived molecules with an important role in various biological processes (physiological or … Copyright © 2021 National Academy of Sciences. It can be envisioned that Ca2+-dependent activation may entail the binding of EF-hand and calmodulin to their respective receiving loops, thereby promoting the NADPH-binding conformation of the nearby residues (Fig. of the plasma membrane (Lambeth, 2002; Panday et al., 2015). S1B). The line connecting their iron atoms is almost exactly perpendicular to the plane of the bilayer (Fig. phox, p67. 46 . Notably, DH residues 515–530 were predicted to interact with loop B (280–292) of TM. No crystal structure data of NOX enzymes are presently available. Human isoforms of the catalytic component of the complex include NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, … Proteases inhibitors (1 μM leupeptine, 1 μM pepstatine, and 1 mM PMSF) were added before cell disruption by Emulsiflex C3 (Avestin) and centrifuged at 60,000 × g for 30 min. The membrane-localized part of the enzyme, referred to as cytochrome b 558, is a heterodimer of the subunits p22 phox (phox is an acronym for phagocyte oxidase) and gp91 phox (also known as NOX2). 5C). Many features indicate that the cavity-bound water molecule actually occupies the position of the dioxygen substrate. Summary: This gene encodes a member of the NADPH oxidase family of enzymes responsible for the catalytic one-electron transfer of oxygen to generate superoxide or hydrogen peroxide. Dysregulation of these polytopic membrane proteins impacts the redox signaling cascades that control cell proliferation and death. 520–540 nm; SPYRO orange, Ex. The additional PW695LEL of mutant csDH is in black (Trp695 side chain is shown as reference). phox . 3 A–C) and exposed toward the external milieu, highlighting the oxygen-reacting center. UV/visible absorption spectra of native and reduced csTM domain support the hexa-coordinated nature of heme binding to NOX5. csDH is depicted in orange with the calmodulin-binding region in blue (R644-V663) (see B), the unstructured EF-hand binding loop in dotted gray (D611-T634), and the protruding hairpin of the FAD-binding lobe in purple (Q489-G509).  |  Secondary structure segments, gathered from the csDH and csTM crystal structures, are indicated. With the insight gained from the individual DH and TM domains, we next addressed the issue of their assembly to model the NOX catalytic core. After this initial discovery, it was found that mammals contain several enzyme isoforms: NOX1–5 and Duox1–2, which differ with respect to their specific activities and tissue distribution (2). Exp Ther Med. (A–C) Overall structure of csTM depicted in different orientations (outer, side, and cytosolic view). Overexpression of csTM was induced by the addition of 0.4 mM IPTG and the temperature was shifted to 17 °C for 16 h. Cells were harvested by centrifugation, resuspended in lysis buffer [50 mM Hepes pH 7.5, 300 mM NaCl, 5% (vol/vol) glycerol], and supplemented with protease inhibitors (1 μM leupeptine, 1 μM pepstatine, and 1 mM PMSF). Various NADPH oxidase components are found in the kidney and glomerular cells. NLM The contour level is 1.3 σ. Indeed, in silico docking shows that upon displacement of Trp695, NADPH can easily be modeled to fit in the crevice at the interface of the NADPH- and FAD-binding lobes of DH with the same binding mode observed across the ferredoxin-NADPH reductase superfamily (Fig. S1C). 31. Electron flow trajectory was calculated with VMD Pathways1.1 plug-in (43). NOX is a multisubunit complex composed of membrane associated subunits of gp91. This segment is longer in human NOX5 than in the other NOX members and binds Hsp90, which is involved in NOX5 stability and activity (17). Melting temperatures for different variants of csTM. (B) A distance of 19.8 Å separates the metal centers of the two heme groups of TM. The observed rates were 44 and 3 s−1 for H317R and 80 and 7 s−1 for R256S. All these NAD+, NADH and NADPH are important co-factors in biological reactions. The catalytic subunit of the enzyme gp91. phox. Free hemin (in 100% DMSO from Sigma) was mixed with csTM and the sample applied to a Superdex-200 (GE Healthcare) equilibrated in storage buffer [50 mM Hepes pH 7.5, 100 mM NaCl, 5% (vol/vol) glycerol, 0.03% (wt/vol) DDM]. In the csDH structure, the EF-hand binding loop is unstructured (D611-T634), probably because of a dynamic role and associated conformational changes that may accompany the enzyme activation. The fluorescence intensity (vertical axis) is plotted against the temperature (horizontal axis). Data collection, phasing and refinement statistics for MAD/SAD structures. COVID-19 is an emerging, rapidly evolving situation. 2004 Jun 18;279(25):26378-86. doi: 10.1074/jbc.M309724200. The principal subunits are shown, together with some of the key cellular and molecular modifications that arise following activation of stress pathways and the generation of superoxide (O2–). contributed equally to this work. The NADPH-oxidases (NOXs) form the only known enzyme family whose sole function is reactive oxygen species (ROS) generation (1, 2). This observation was fully consistent with published peptide-binding experiments on human NOX2 and NOX4, which identified the residues homologous to 507–533 of csNOX as the B-loop binding region (31) (Fig. and cytosolic subunits of p47. Purified recombinant C. stagnale DH (residues 413–693; csDH) did not retain the FAD cofactor, possibly a symptom of poor protein stability, and crystals did not grow in any of the tested conditions. csTM was concentrated to 25 mg/mL and mixed with monoolein (1-oleoyl-rac-glycerol) in a 2:3 protein to lipid ratio (wt/wt) using two coupled syringes (Hamilton) at 20 °C. Moreover, upon extraction from the membranes, these enzymes tend to proteolyze spontaneously and lose their noncovalently bound cofactors (FAD and hemes). Our findings indicate avenues for the pharmacological manipulation of NOX activity. Published online 2007 Aug 31. doi: 10.1016/j.freeradbiomed.2007.08.014 Characterization of the mutant csDH domain and its structure in complex with FAD. 300 μM of FAD were added to WT DH and Phe693 mutants. Role of the Rho GTPase Rac in the activation of the phagocyte NADPH oxidase: outsourcing a key task. The NADPH oxidase of professional phagocytes is a crucial component of the innate immune response due to its fundamental role in the production of reactive oxygen species that act as powerful microbicidal agents. Its H-bonding environment is clearly suited for O2 binding and sequestration (Fig. The protein absorbance spectrum was fully consistent with csTM containing two bound heme groups. Whereas NOX5 and DUOX contain one non-canonical and three canonical EF-hand motifs, the regulatory domain of OsRBOH-B has only one functional EF-hand motif followed by one disabled EF-hand and two EF-like motifs. Fluorescent labeling of the leukocyte NADPH oxidase subunit p47(phox): evidence for amphiphile-induced conformational changes. In close proximity to the toggle switch Phe693, there is the positively charged Lys361, which is an Arg in the other NOXs, except for the constitutively active NOX4 where there is a Val (Fig. 2 and Fig. The gene encoding for Cylindrospermum stagnale NOX5 was purchased from GeneScript. NADH is the reduced form of NAD+. In agreement with the notion that the site lined by Arg256 and His317 is involved in O2 binding and catalysis, we found that reoxidation of chemically reduced csTM is greatly impaired by mutations targeting these two residues (Fig. The melting curve was built by measuring the height of the elution peak at 414 nm for each temperature and data were analyzed by nonlinear regression using Prism software (GraphPad). By investigating the individual catalytic domains, we elucidate the core of the NOX 3D structure. Researchers are still trying to understand what causes this strong correlation between neural and social networks. Astronomers thought they’d finally figured out where gold and other heavy elements in the universe came from. Phe693, at the C terminus, is in dark gray. Epub 2020 Jul 1. The sample was concentrated using an Amicon concentrator with a 10-kDa cut-off and loaded on a desalting column (GE Healthcare) equilibrated in low salt buffer [LSbuffer; 50 mM Tris⋅HCl pH 7.5, 5% (vol/vol) glycerol, 50 mM NaCl]. Regulates signaling cascades probably through phosphatases inhibition. NADPH oxidase structure.NADPH oxidase is a multi-subunit enzyme complex present in the vesicular and plasma membranes of leukocytes. CRISPR-Cas9 gene editing can improve the effectiveness of spermatogonial stem cell transplantation in mice and livestock, a study finds. Although the evidence is still limited, a growing body of research suggests music may have beneficial effects for diseases such as Parkinson’s. The initial phases were obtained by iron-based single-wavelength anomalous dispersion using the program autoSHARP (40). We describe the atomic crystal structures of the catalytic flavin adenine dinucleotide (FAD)- and heme-binding domains of Cylindrospermum stagnale NOX5. The reaction becomes fourfold faster using ferricyanide as electron acceptor (kcat = 261.9 ± 27.7 min−1 and Km = 84.54 ± 22.8 μM). and p22. The in meso mix was dispensed manually using a Hamilton syringe coupled to a repetitive dispenser onto a sandwich plate in a 120-nL bolus overlaid by 1 μL of precipitant solution. The anaerobic reaction of csTM enzymes with sodium dithionite in a sealed vial yielded completely reduced enzyme only when the sodium dithionite/enzyme ratio was higher than stoichiometric. A consensus regarding basic features of the core region of NOX enzymes has emerged based on indirect data. Author contributions: F.M., S.N., and A.M. designed research; F.M., S.N., E.M.F., M.C., E.R., and M.W.F. (29). S1E). A model for the TM–DH core of NOX catalytic subunit as gathered from the crystal structures of DH and TM domains from csNOX5. Of relevance, the catalytic subunits of the oligomeric NOX1–4 also consist only of DH–TM with no other domains (14). 2012 Jul;69(14):2283-305. doi: 10.1007/s00018-012-1007-4. S3 and Table S3). (A) The in silico docking of csTM (light blue) and csDH (orange) structures is shown in a putative active conformation (Fig. 2002 Mar 8;277(10):8421-32. doi: 10.1074/jbc.M109778200. DH contains the binding sites for FAD (flavin adenine dinucleotide) and NADPH, whereas TM binds two hemes (1, 2, 13). The 2.0-Å resolution crystal structure of csTM has an overall pyramidal shape with a triangular base on the inner membrane side and a narrower apex toward the outer membrane face (Table S1). The metal-to-metal distance is 19.8 Å, whereas the shortest interatomic distance (6.4 Å) is between vinyl 2 of the inner heme and vinyl 4 of the outer heme (Fischer nomenclature). 1A). A high reoxidation rate for WT csTM was estimated by fitting the stopped-flow trace at 427 nm to a single-exponential function (∼300 s−1). S6). Solubilization proceeded for 2 h. The solubilized sample was centrifuged and the supernatant was loaded onto a TALON resin. For anaerobiosis experiments, a sealed cuvette was used under Argon flow. Specifically, Asn288 (B-loop of TM), Lys361 (D-loop of TM), Thr520 (B-loop binding region of DH) (31), and Phe693 (flavin-interacting C terminus of DH) were defined as “active” residues: that is, directly involved in the domain–domain interactions. A first visual analysis immediately indicated that the 3D structures of the two domains could be easily docked with the outer heme and the flavin within 5 Å distance from each other and C-terminal Lys412 of TM linked to N-terminal Glu413 of DH. TM of NOX consists of six transmembrane helices and contains two heme groups positioned almost orthogonally to the lipid bilayer. It is instead an outersphere reaction that affords reduction of molecular oxygen through an electron transfer step, as originally suggested by Isogai et al. Nicotinamide Adenine Dinucleotide (NAD+) is a coenzyme present in biological systems. In NOX4 and NOX2, these loops were shown to contribute to the regulation of the enzyme activity (31, 32). Solutions (2–3 mL) were prepared in glass vials (5 mL) sealed with a screw-cap with hole and PTFE/silicone septum. Although calmodulin is not found in prokaryotic cells, the conservation of the calmodulin-binding region (37% identity between human and C. stagnale) (Fig. Structure of NADPH oxidase The structure of NADPH oxidase is quite complex, consisting of two membrane-bound elements (gp91 PHOX and p22 PHOX), three cytosolic components (p67 PHOX, p47 PHOX and p40 PHOX), and a low-molecular-weight G protein (either rac 2 or rac 1). The model was further improved with iterative cycles of coot, phenix.fem and Refmac5 (38, 39). Transmembrane helices are labeled sequentially as h1–h6. Characteristic structural features of csDH. This geometry is obviously suited to promote the interdomain electron transfer that injects the NADPH-donated electrons from the flavin to the heme-Trp378-heme array. Epub 2020 May 26. The NOX family of ROS-generating NADPH oxidases: Physiology and pathophysiology, Nox enzymes and new thinking on reactive oxygen: A double-edged sword revisited, Genetic disorders coupled to ROS deficiency, Nox/Duox family of NADPH oxidases: Lessons from knockout mouse models, Combating oxidative stress in vascular disease: NADPH oxidases as therapeutic targets, NADPH oxidase 4 (Nox4) is a major source of oxidative stress in the failing heart, NADPH oxidases: Novel therapeutic targets for neurodegenerative diseases, Compound heterozygosity for a novel hemizygous missense mutation and a partial deletion affecting the catalytic core of the H, Aiding and abetting roles of NOX oxidases in cellular transformation, ROS-generating NADPH oxidase NOX4 is a critical mediator in oncogenic H-Ras-induced DNA damage and subsequent senescence, Oncogene-induced reactive oxygen species fuel hyperproliferation and DNA damage response activation, Therapeutic potential of NADPH oxidase 1/4 inhibitors, Intramembrane bis-heme motif for transmembrane electron transport conserved in a yeast iron reductase and the human NADPH oxidase, Evolution of the ferric reductase domain (FRD) superfamily: Modularity, functional diversification, and signature motifs, Homologs of gp91phox: Cloning and tissue expression of Nox3, Nox4, and Nox5, Nox5 stability and superoxide production is regulated by C-terminal binding of Hsp90 and CO-chaperones, Identification of the binding site for the regulatory calcium-binding domain in the catalytic domain of NOX5, NADPH oxidase 5 (NOX5) interacts with and is regulated by calmodulin, A comprehensive review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes, Studies on neutrophil b-type cytochrome in situ by low temperature absorption spectroscopy, EPR signals of cytochrome b558 purified from porcine neutrophils, To be or not to be an oxidase: Challenging the oxygen reactivity of flavoenzymes, Superoxide dismutase from the eukaryotic thermophile, A variant X-linked chronic granulomatous disease patient (X91+) with partially functional cytochrome b, X-Linked chronic granulomatous disease: Mutations in the CYBB gene encoding the gp91-phox component of respiratory-burst oxidase, Role of putative second transmembrane region of Nox2 protein in the structural stability and electron transfer of the phagocytic NADPH oxidase, The molecular mechanism of autoxidation for myoglobin and hemoglobin: A venerable puzzle, The mechanism of electron donation to molecular oxygen by phagocytic cytochrome b558, Nox5 and the regulation of cellular function, Nox4 B-loop creates an interface between the transmembrane and dehydrogenase domains, Characterization of superoxide overproduction by the D-Loop(Nox4)-Nox2 cytochrome b(558) in phagocytes—Differential sensitivity to calcium and phosphorylation events, Cytochrome b558: The flavin-binding component of the phagocyte NADPH oxidase, Inhibition of NADPH oxidase activation by peptides mapping within the dehydrogenase region of Nox2-A “peptide walking” study, Defining NADH-driven allostery regulating apoptosis-inducing factor, Overview of the CCP4 suite and current developments, PHENIX: A comprehensive Python-based system for macromolecular structure solution, Coot: Model-building tools for molecular graphics, Automated structure solution with autoSHARP, DM: An automated procedure for phase improvement by density modification, UCSF Chimera—A visualization system for exploratory research and analysis, Exploring biological electron transfer pathway dynamics with the Pathways plugin for VMD, The HADDOCK2.2 Web Server: User-friendly integrative modeling of biomolecular complexes, Prediction of homoprotein and heteroprotein complexes by protein docking and template-based modeling: A CASP-CAPRI experiment, Immunodeficiency mutation databases (IDbases), Proceedings of the National Academy of Sciences, Earth, Atmospheric, and Planetary Sciences, www.pnas.org/lookup/suppl/doi:10.1073/pnas.1702293114/-/DCSupplemental, Crystal structures and atomic model of NADPH oxidase, Science & Culture: At the nexus of music and medicine, some see disease treatments, News Feature: Tracing gold's cosmic origins, Journal Club: Friends appear to share patterns of brain activity, Transplantation of sperm-producing stem cells.

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