5.B.1 The gp91phox Phagocyte NADPH Oxidase-associated Cytochrome b558 (Phox) Family
The human phagocyte cytochrome b558 is a heterodimeric complex consisting of a heavy (β) chain (gp91phox) and a light (α) chain (p22phox) as well as several auxiliary subunits (Geisz and Leto, 2004). The β-chain is a glycoprotein of 570 amino acyl residues called gp91phox, the product of the X-linked chronic granulomatous disease gene. The protein bears (1) the heme-binding site in its N-terminal 280 residues, and (2) an FAD binding site (residues 338-344) as part of the C-terminal NADPH oxidase domain. The N-terminal domain has 6 putative transmembrane spanners (TMSs) and is the cytochrome binding site. It has been reported to catalyze efflux of protons through an H+ channel that acts as a charge compensation pathway for the electrogenic generation of the superoxide radical, O2&149;-. The proposal that (gp91phox) has H+ channel activity has been effectively disputed and is now in doubt (DeCoursey 2003; DeCoursey et al. 2002, 2001, Morgan et al. 2002, 2007). Further, the voltage-gated proton channel (TC# 1.A.51) is expressed on phagosomes (Okochi et al., 2009 ). As a result, this family has been removed from class 1.A where it previously assumed the TC#1.A.20.
gp91phox is the terminal component of a respiratory chain that transfers single electrons from cytoplasmic NADPH to O2 on the external side of the plasma membrane generating superoxide. Its activity is electrogenic, causing depolarization of the membrane potential, negative inside. Four histidines, two in TMS3 (his-101 and his-115) and two in TMS5 (his-209 and his-222), are known to ligand heme (Biberstine-Kinkade et al. 2001). p22phox (the α-chain) is not homologous to anything else in the databases and may not have catalytic activity.
The activity of gp91phox is accompanied by a fall in the internal pH. Efflux of H+ through a channel would be expected to provide charge compensation, preventing a large fall in the internal pH. In the presence of arachidonic acid, the direction of H+ flux is dictated by the pmf. The oxidase interacts with various cytosolic proteins that may regulate its activity. Differential cell surface expression of Nox1, Nox2 and Nox5 (for example) is accomplished via two pathways, a Sar1 (small GTPase)-dependent pathway and a Sar-independent pathway (Kiyohara et al. 2018).
gp91phox is homologous throughout its length to proteins from animals, slime molds, plants, fungi and bacteria. There are seven homologues in humans (Nox1-5 and Duox1 and 2). Homologues in all organisms form six phylogenetic clusters (Kimball and Saier, 2002). These include mitogenic oxidases of animals, the so-called respiratory burst oxidase proteins of plants, and the ferric reductases of yeast and plants. In all cases, electrons are probably transferred across the membrane (from in to out) forming superoxide. Sixteen homologues of the Phox family have been sequenced from A. thaliana. Homologues are also found in C. elegans and D. melanogaster.
The overall electron transfer reaction catalyzed by gp91phox and some of its homologues is:
1. electron (in) → electron (out)
2. O2 (out) + electron (in) → O2- (superoxide) (out)
This family belongs to the Calmodulin/Calcineurin/KChIP Superfamily.
|Biberstine-Kinkade, K.J., F.R. DeLeo, R.I. Epstein, B.A. LeRoy, W.M. Nauseef, and M.C. Dinauer. (2001). Heme-ligating histidines in flavocytochrome b(558): identification of specific histidines in gp91phox. J. Biol. Chem. 276: 31105-31112.|
|Cheng, G., Z. Cao, X. Xu, E.G. van Meir, and J.D. Lambeth. (2001). Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5. Gene 269: 131-140.|
|Connolly, E.L., N.H. Campbell, N. Grotz, C.L. Prichard, and M.L. Guerinot. (2003). Overexpression of the FRO2 ferric chelate reductase confers tolerance to growth on low iron and uncovers posttranscriptional control. Plant Physiol. 133: 1102-1110.|
|Dancis, A., D.G. Roman, G.J. Anderson, A.G. Hinnebusch, and R.D. Klausner. (1992). Ferric reductase of Saccharomyces cerevisiae: molecular characterization, role in iron uptake, and transcriptional control by iron. Proc. Natl. Acad. Sci. USA 89: 3869-3873.|
|DeCoursey T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M. C. Dinauer. (2001). The gp91phox component of NADPH oxidase is not the voltage-gated proton channel in phagocytes, but it helps. J. Biol. Chem. 276: 36063-36066.|
|DeCoursey T.E., V.V. Cherny, W. Zhou, and L.L. Thomas. (2000). Simultaneous activation of NADPH oxidase-related proton and electron currents in human neutrophils. PNAS. 97: 6885-6889.|
|DeCoursey, T.E. (2003). Interactions between NADPH oxidase and voltage-gated proton channels: why electron transport depends on proton transport. FEBS Lett. 555: 57-61.|
|DeCoursey, T.E., D. Morgan, and V.V. Cherny. (2002). The gp91phox component of NADPH oxidase is not a voltage-gated proton channel. J Gen Physiol 120: 773-779.|
|Desikan, R., K. Last, R. Harrett-Williams, C. Tagliavia, K. Harter, R. Hooley, J.T. Hancock, and S.J. Neill. (2006). Ethylene-induced stomatal closure in Arabidopsis occurs via AtrbohF-mediated hydrogen peroxide synthesis. Plant J. 47: 907-916.|
|Dinauer, M.C., S.N. Orkin, and R. Brown (1987). The glycoprotein encoded by the X-linked chronic granulomatous disease locus is a component of the neutrophil cytochrome b complex. Nature 327: 717-720.|
|Einset, J., P. Winge, A.M. Bones, and E.L. Connolly. (2008). The FRO2 ferric reductase is required for glycine betaine''s effect on chilling tolerance in Arabidopsis roots. Physiol Plant 134: 334-341.|
|Enomoto, Y., H. Hodoshima, H. Shimada, K. Shoji, T. Yoshihara, and F. Goto. (2007). Long-distance signals positively regulate the expression of iron uptake genes in tobacco roots. Planta. 227(1):81-89.|
|Finegold, A.A., K.P. Shatwell , A.W. Segal, R.D. Klausner and A. Dancis (1996). Intramembrane bis-heme motif for transmembrane electron transport conserved in a yeast iron reductase and the human NADPH oxidase. J. Biol. Chem. 271: 31021-31024.|
|Fisher, A.B. (2009). Redox signaling across cell membranes. Antioxid Redox Signal 11: 1349-1356.|
|Geiszt, M, and T.L. Leto. (2004). The Nox family of NAD(P)H oxidases: host defense and beyond. J. Biol. Chem. 279: 51715-51718. |
|Georgatsou, E., L.A. Mavrogiannis, G.S. Fragiadakis, and D. Alexandraki. (1997). The yeast Fre1p/Fre2p cupric reductases facilitate copper uptake and are regulated by the copper-modulated Mac1p activator. J. Biol. Chem. 272: 13786-13792.|
|Hajjar, C., M.V. Cherrier, G. Dias Mirandela, I. Petit-Hartlein, M.J. Stasia, J.C. Fontecilla-Camps, F. Fieschi, and J. Dupuy. (2017). The NOX Family of Proteins Is Also Present in Bacteria. MBio 8:.|
|Han, O., V.P. Miller, and H.W. Liu. (1990). Mechanistic studies of the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis. Purification and characterization of CDP-6-deoxy-delta 3,4-glucoseen reductase based on its NADH:dichlorophenolindolphenol oxidoreductase activity. J. Biol. Chem. 265: 8033-8041.|
|Hassett, R. and D.J. Kosman. (1995). Evidence for Cu(II) reduction as a component of copper uptake by Saccharomyces cerevisiae. J. Biol. Chem. 270: 128-134.|
|Henderson, L.M. (1998). Role of histidines identified by mutagenesis in the NADPH oxidase-associated H+ channel. J. Biol. Chem. 273: 33216-33223.
|Henderson, L.M., S. Thomas, G. Banting, and J.B. Chappell (1997). The arachidonate-activatable, NADPH oxidase-associated H+ channel is contained within the multi-membrane-spanning N-terminal region of gp91-phox. Biochem. J. 325: 701-705.
|Hovind, L.J., M.R. Skerritt, and D.L. Campbell. (2011). K(V)4.3 N-terminal deletion mutant Δ2-39: effects on inactivation and recovery characteristics in both the absence and presence of KChIP2b. Channels (Austin) 5: 43-55.|
|Jerng, H.H. and P.J. Pfaffinger. (2014). Modulatory mechanisms and multiple functions of somatodendritic A-type K (+) channel auxiliary subunits. Front Cell Neurosci 8: 82.|
|Kimball R.A., M.H. Saier. (2002).Voltage-gated H+ channels associated with human phagocyte superoxide-generating NADPH oxidases: sequence comparisons, structural predictions, and phylogenetic analyses. Mol. Membr. Biol. 19:137-47.|
|Kiyohara, T., K. Miyano, S. Kamakura, J. Hayase, K. Chishiki, A. Kohda, and H. Sumimoto. (2018). Differential cell surface recruitment of the superoxide-producing NADPH oxidases Nox1, Nox2 and Nox5: The role of the small GTPase Sar1. Genes Cells 23: 480-493.|
|Kwak, J.M., I.C. Mori, Z.M. Pei, N. Leonhardt, M.A. Torres, J.L. Dangl, R.E. Bloom, S. Bodde, J.D. Jones, and J.I. Schroeder. (2003). NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis. EMBO. J. 22: 2623-2633.|
|Lambeth J.D., G. Cheng, R.S. Arnold and W.A. Edens (2000). Novel homologs of gp91phox. Trends Biochem. Sci. 25: 459-461.
|Lesuisse, E., M. Casteras-Simon, and P. Labbe. (1996). Evidence for the Saccharomyces cerevisiae ferrireductase system being a multicomponent electron transport chain. J. Biol. Chem. 271: 13578-13583.|
|Lin, H., W. Du, Y. Yang, K.S. Schumaker, and Y. Guo. (2014). A calcium-independent activation of the Arabidopsis SOS2-like protein kinase24 by its interacting SOS3-like calcium binding protein1. Plant Physiol. 164: 2197-2206.|
|Morgan D., V.V. Cherny, A.F. Finnegan, J. Bollinger, M.H. Gelb, and T.E. DeCoursey. (2007). Sustained activation of proton channels and NADPH oxidase in human eosinophils and murine granulocytes requires PKC but not cPLA2α activity. J. Physiol 579.2: 327-344.|
|Morgan, D., V.V. Cherny, M.O. Price, M.C. Dinauer, and T.E. DeCoursey. (2002). Absence of proton channels in COS-7 cells expressing functional NADPH oxidase components. J Gen Physiol 119: 571-580.|
|O''Neill, S., M. Mathis, L. Kovacic, S. Zhang, J. Reinhardt, D. Scholz, U. Schopfer, R. Bouhelal, and U.G. Knaus. (2018). Quantitative interaction analysis permits molecular insights into functional NOX4 NADPH oxidase heterodimer assembly. J. Biol. Chem. [Epub: Ahead of Print]|
|Okochi, Y., M. Sasaki, H. Iwasaki, and Y. Okamura. (2009). Voltage-gated proton channel is expressed on phagosomes. Biochem. Biophys. Res. Commun. 382: 274-279.|
|Paffenholz, R., R.A. Bergstrom, F. Pasutto, P. Wabnitz, R.J. Munroe, W. Jagla, U. Heinzmann, A. Marquardt, A. Bareiss, J. Laufs, A. Russ, G. Stumm, J.C. Schimenti, and D.E. Bergstrom. (2004). Vestibular defects in head-tilt mice result from mutations in Nox3, encoding an NADPH oxidase. Genes Dev. 18: 486-491.|
|Picciocchi, A., F. Debeurme, S. Beaumel, M.C. Dagher, D. Grunwald, A.J. Jesaitis, and M.J. Stasia. (2011). Role of putative second transmembrane region of Nox2 protein in the structural stability and electron transfer of the phagocytic NADPH oxidase. J. Biol. Chem. 286: 28357-28369.|
|Rees, E.M. and D.J. Thiele. (2007). Identification of a vacuole-associated metalloreductase and its role in Ctr2-mediated intracellular copper mobilization. J. Biol. Chem. 282: 21629-21638.|
|Rehman, L., X. Su, X. Li, X. Qi, H. Guo, and H. Cheng. (2017). FreB is involved in the ferric metabolism and multiple pathogenicity-related traits of Verticillium dahliae. Curr. Genet. [Epub: Ahead of Print]|
|Robinson, N.J., C.M. Procter, E.L. Connolly, and M.L. Guerinot. (1999). A ferric-chelate reductase for iron uptake from soils. Nature 397: 694-697.|
|Shatwell, K.P., A. Dancis, A.R. Cross, R.D. Klausner and A.W. Segal (1996). The FRE1 ferric reductase of Saccharomyces cerevisiae is a cytochrome b similar to that of NADPH oxidase. J. Biol. Chem. 271: 14240-14244.|
|Shi, X., C. Stoj, A. Romeo, D.J. Kosman, and Z. Zhu. (2003). Fre1p Cu2+ reduction and Fet3p Cu1+ oxidation modulate copper toxicity in Saccharomyces cerevisiae. J. Biol. Chem. 278: 50309-50315.|
|Song, C.J., I. Steinebrunner, X. Wang, S.C. Stout, and S.J. Roux. (2006). Extracellular ATP induces the accumulation of superoxide via NADPH oxidases in Arabidopsis. Plant Physiol. 140: 1222-1232.|
|Thomsen, M.B., C. Wang, N. Ozgen, H.G. Wang, M.R. Rosen, and G.S. Pitt. (2009). Accessory subunit KChIP2 modulates the cardiac L-type calcium current. Circ Res 104: 1382-1389.|
|Torres, M.A., H. Onouchi, S. Hamada, C. Machida, K.E. Hammond-Kosack, J.D. Jones (1998). Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91phox). Plant J. 14: 365-370.
|Wang, H.G., X.P. He, Q. Li, R.D. Madison, S.D. Moore, J.O. McNamara, and G.S. Pitt. (2013). The auxiliary subunit KChIP2 is an essential regulator of homeostatic excitability. J. Biol. Chem. 288: 13258-13268.|
|Wu, H., L. Li, J. Du, Y. Yuan, X. Cheng, and H.Q. Ling. (2005). Molecular and biochemical characterization of the Fe(III) chelate reductase gene family in Arabidopsis thaliana. Plant Cell Physiol. 46: 1505-1514.|
|Yu, H., Z. Peng, Y. Zhan, J. Wang, Y. Yan, M. Chen, W. Lu, S. Ping, W. Zhang, Z. Zhao, S. Li, M. Takeo, and M. Lin. (2011). Novel regulator MphX represses activation of phenol hydroxylase genes caused by a XylR/DmpR-type regulator MphR in Acinetobacter calcoaceticus. PLoS One 6: e17350.|
|Yun, C.W., M. Bauler, R.E. Moore, P.E. Klebba, and C.C. Philpott. (2001). The role of the FRE family of plasma membrane reductases in the uptake of siderophore-iron in Saccharomyces cerevisiae. J. Biol. Chem. 276: 10218-10223.|
The gp91phox/p22phox NADPH oxidase-associated, cytochrome b558, Nox2. TMS2 is important for stability and electron transfer (Picciocchi et al., 2011). The integral membrane flavocytochrome of Nox 2 transfers an electron from
intracellular NADPH to extracellular O2, generating superoxide anion, O2- (Fisher 2009).
gp91phox (β-chain) of Homo sapiens (Nox2) (P04839)
p22phox (α-chain) of Homo sapiens (P13498)
Nucleus/kidney/muscle/endothelial cell superoxide-generating NADPH oxidase (Nox4) (may regulate gene expression) (Cheng et al., 2001; Kuroda et al., 2005). Integrated analyses of heterodimerization, trafficking and catalytic activity have identified determinants for the NOX4-p22phox interaction such as heme incorporation into NOX4 and hot spot residues in TMSs 1 and 4 in p22phox; their effects on NOX4 maturation and ROS generation were analyzed (O'Neill et al. 2018).
Animals and slime molds
Kidney superoxide-generating NADPH oxidase of Homo sapiens
|5.B.1.1.3||Multiple tissue mitogenic oxidase, subunit 65 (Mox1 or Nox1) (alternative splicing yields a 191 aa protein with H+ channel activity) (Bánfi et al., 2000; Suh et al., 1999)||Animals and slime molds||Mitogenic oxidase (Nox1) of Homo sapiens |
|5.B.1.1.4||Mitogenic NADPH oxidase 3, Nox3 (Cheng et al., 2001). Critical for formation of otoconia, mineral crystals in the inner ear; mutants are defective for motion and gravity sensing (Paffenholz et al., 2004).||Animals||Nox3 of Homo sapiens (Q9HBY0)|
|5.B.1.1.5||NADPH oxidase 5 (Nox5) (Cheng et al., 2001)||Animals||Nox5 of Homo sapiens (Q96PH1)|
|5.B.1.1.6||Thyroid NADPH oxidase/peroxidase 1 (Dual oxidase 1; Duox1) with two EF band domains, responsive to Ca2+ regulation (De Deken et al., 2000; Edens et al., 2001)||Animals||Duox1 of Homo sapiens (Q9NRD9)|
|5.B.1.1.7||Thyroid NADPH oxidase/peroxidase 2 (Dual oxidase 2; Duox2) with two EF band domains, responsive to Ca2+ regulation (De Deken et al., 2000; Edens et al., 2001)||Animals||Duox2 of Homo sapiens (Q9NRD8)|
|5.B.1.1.8||Respiratory burst oxidase proteins A-F||Plants||Respiratory burst oxidase A of Arabidopsis thaliana|
Respiratory burst oxidase homologue F, RBOHF, of 944 aas and 6 TMSs. Calcium-dependent NADPH oxidase that
generates superoxide. Generates reactive oxygen species (ROS) during
incompatible interactions with pathogens and is important in the
regulation of the hypersensitive response (HR). Involved in abscisic
acid-induced stomatal closing and in UV-B and abscisic acid
ROS-dependent signaling (Song et al. 2006; Desikan et al. 2006; Kwak et al. 2003).
RBOHF of Arabidopsis thaliana (Mouse-ear cress)
NADPH oxido-reductase of 450 aas and 7 TMSs. These proteins have been characterized in prokaryotes as well as eukaryotes (Hajjar et al. 2017).
Putative oxido-reductase of Vibrio cholerae
Uncharacterized oxidoreductase of 445 aas and 6 N-terminal TMSs.
Putative oxidoreductase of Streptomyces sp.
Ferric reductase like transmembrane component of 220 aas and 6 TMSs.
Ferric reductase of Thermophagus xiamenensis
Uncharacterized protein of 441 aas and 6 N-terminal TMSs.
UP of Planctomycetes bacterium
Uncharacterized protein of 215 aas and 6 TMSs.
UP of Candidatus Curtissbacteria bacterium
Uncharacterized protein of 205 aas and 6 TMSs.
UP of Candidatus Saccharibacteria bacterium
Uncharacterized protein of 210 aas and 6 TMSs.
UP of Candidatus Nomurabacteria bacterium
Uncharacterized protein of 222 aas and 6 TMSs.
UP of Alicyclobacillus sendaiensis
Uncharacterized ferric reductase domain protein transmembrane component of 287 aas and 6 TMSs.
UP of Candidatus Moranbacteria bacterium
CDP-6-deoxy-delta-3,4-glucoseen reductase (Han et al. 1990).
CDP-6-deoxy-delta-3,4-glucoseen reductase of Yersinia pseudotuberculosis
Phenol hydroxylase (Yu et al. 2011).
Phenol hydroxylase of Acinetobacter calcoaceticus
|5.B.1.4.1||Ferric reductase/oxidase, FRO1||Plants||FRO1 of Arabidopsis thaliana|
|5.B.1.4.2||Iron chelate reductase, Fox1 (La Fontaine et al., 2002)||Algae||Fox1 of Chlamydomonas reinhardtii (ABM66085)|
|5.B.1.4.3||Fro1, ferric chelate reductase (Enomoto et al., 2007).||Plants||Fro1 of Lycopersicon esculentum (Q6EMC0)|
Ferric reduction oxidase 2, Fro2 or Frd1, of 725 aas. Flavocytochrome that transfers electrons
across the plasma membrane to reduce ferric iron chelates and form
soluble ferrous iron in the rhizosphere. May be involved in the delivery
of iron to developing pollen grains. Acts also as a copper-chelate
reductase. Involved in glycine betaine-mediated chilling tolerance and
reactive oxygen species accumulation (Robinson et al. 1999; Wu et al. 2005; Connolly et al. 2003; Einset et al. 2008).
Fro2 of Arabidopsis thaliana (Mouse-ear cress)
Plasma membrane Fe3+ and Cu2+ reductase, Fre1 (transfers electrons from NADPH in the cytoplasm to Fe3+ and Cu2+ in the extracellular millieu) (Rees and Thiele, 2007). Thus, it, as well as Fre2 (TC# 5.B.1.7.2), mediate the reductive uptake of Fe3+-salts and Fe3+ bound to catecholate or hydroxamate siderophores. Fe3+ is reduced to Fe2+, which then dissociates from the siderophore and can be imported by the high-affinity Fe2+ transport complex in the plasma membrane. Also participates in Cu2+ reduction and Cu+ uptake (Dancis et al. 1992; Hassett and Kosman 1995; Lesuisse et al. 1996; Georgatsou et al. 1997; Shi et al. 2003).
Fre1 of Saccharomyces cerevisiae
|5.B.1.5.2||Vacuolar Fe3+ and Cu2+ reductase, Fre6 (transfers electrons from NADPH in the cytoplasm to Cu2+ in the vacuole) (Rees and Thiele, 2007). ||Yeast ||Fre6 of Saccharomyces cerevisiae|
Fre2 Fe3+/Cu2+ oxidoreductase of 711 aas; similar in catalytic function to Fre1 (TC# 5.B.1.5.1), but induced only by Fe3+, not Cu2+ (Georgatsou et al. 1997; Yun et al. 2001).
Fre2 of Saccharomyces cerevisiae
Ferric reductase, FreB of 582 aas and 7 TMSs (Rehman et al. 2017).
FreB of Verticillium dahliae (Verticillium wilt)