Abstract
All members of the NOX protein family contain a unique b-type cytochrome that mediates the electron transport that characterizes the activity of the multicomponent oxidase complexes. Referred to as cytochrome b558, because of its signature spectral absorbance at 558 nm in reduced-minus-oxidized difference spectroscopy, or cytochrome b(-245), because of its very low midpoint potential of −245 mV at pH 7.0, the protein possesses two stacked inequivalent hemes ligated by pairs of histidine residues in membrane helices h3 and h5. In a flavin-dependent fashion, cytochrome b558 shuttles electrons from cytoplasmic NADPH across membranes to molecular oxygen and thereby generates superoxide anion. By performing reduced-minus-oxidized difference spectroscopy and using the millimolar extinction coefficient, E 559–540 nm = 21.6 cm−1 mM−1, one can calculate the amount of cytochrome b558 in intact cells or partially purified membrane preparations. Measurements in samples where cytochrome b558 is relatively high and the presence of unrelated heme-containing proteins low, as in neutrophils, are straightforward. However, low levels of cytochrome b558 expression combined with an abundance of mitochondria and other sources of heme proteins make spectral detection of cytochrome b558 in non-phagocytic cells extremely challenging.
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References
Biberstine-Kinkade KJ, DeLeo FR, Epstein RI, LeRoy BA, Nauseef WM, Dinauer MC (2001) Heme-ligating histidines in flavocytochrome b 558. J Biol Chem 276:31105–31112
Magnani F, Nenci S, Millana Fananas E, Ceccon M, Romero E, Fraaije MW, Mattevi A (2017) Crystal structures and atomic model of NADPH oxidase. Proc Natl Acad Sci U S A 114(26):6764–6769. https://doi.org/10.1073/pnas.1702293114
Baldridge CW, Gerard RW (1933) The extra respiration of phagocytosis. Am J Phys 103:235–236
Sbarra AJ, Karnovsky ML (1959) The biochemical basis of phagocytosis. I. Metabolic changes during the ingestion of particles by polymorphonuclear leukocytes. J Biol Chem 234:1355–1362
Iyer GYN, Islam DMF, Quastel JH (1961) Biochemical aspects of phagocytosis. Nature 192:535–541
Hattori H (1961) Studies on the labile, stable Nadi oxidase and peroxidase staining reactions in the isolated particles of horse granulocyte. Nagoya J Med Sci 23:362–378
Shinagawa Y, Tanaka C, Teroaka A, Shinagawa Y (1966) A new cytochrome in neutrophilic granules of rabbit leucocyte. J Biochem 59:622–624
Shinagawa Y, Tanaka C, Teroaka A (1966) Electron microscopic and biochemical study of the neutrophilic granules from leucocytes. J Electron Microsc 15:81–85
Segal AW, Jones OTG (1978) Novel cytochrome b system in phagocytic vacuoles of human granulocytes. Nature 276:515–517
Berendes H, Bridges RA, Good RA (1957) A fatal granulomatosus of childhood; the clinical study of a new syndrome. Minn Med 40(5):309–312
Segal AW, Jones OT, Webster D, Allison AC (1978) Absence of a newly described cytochrome b from neutrophils of patients with chronic granulomatous disease. Lancet 2:446–449
Cross AR, Jones OTG, Harper AM, Segal AW (1981) Oxidation-reduction properties of the cytochrome b found in the plasma-membrane fraction of human neutrophils. A possible oxidase in the respiratory burst. Biochem J 194:599–606
Cross AR, Higson FK, Jones OTG (1982) The enzymic reduction and kinetics of oxidation of cytochrome b of neutrophils. Biochem J 204:479–485
Cross AR, Jones OTG, Garcia R, Segal AW (1982) The association of FAD with the cytochrome b-245 of human neutrophils. Biochem J 208:759–763
Cross AR, Rae J, Curnutte JT (1995) Cytochrome b-245 of the neutrophil superoxide-generating system contains two nonidentical hemes. Potentiometric studies of a mutant form of gp91phox. J Biol Chem 270:17075–17077
Light DR, Walsh C, O’Callaghan AM, Goetzl EJ, Tauber AI (1981) Characteristics of the cofactor requirements for the superoxide-generating NADPH oxidase of human polymorphonuclear leukocytes. Biochemistry 20:1468–1476
Cross AR, Jones OT (1991) Enzymic mechanisms of superoxide production. Biochim Biophys Acta 1057(3):281–298
Bedard K, Krause KH (2007) The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 87:245–313
Bedard K, Lardy B, Krause KH (2007) NOX family NADPH oxidases: not just in mammals. Biochemie 89:1107–1112
Aguirre J, Lambeth JD (2010) Nox enzymes from fungus to fly to fish and what they tell us about nox function in mammals. Free Radic Biol Med 49:1342–1353
Nakamura M, Murakami M, Koga T, Tanaka Y, Minakami S (1987) Monoclonal antibody 7D5 raised to cytochrome b 558 of human neutrophils: immunocytochemical detection of the antigen in peripheral phagocytes of normal subjects, patients with chronic granulomatous disease, and their carrier mothers. Blood 69(5):1404–1408
Burritt JB, DeLeo FR, McDonald CL, Prigge JR, Dinauer MC, Nakamura M, Nauseef WM, Jesaitis AJ (2001) Phage display epitope mapping of human neutrophil flavocytochrome b558. Identification of two juxtaposed extracellular domains. J Biol Chem 276:2053–2061
Radeke HH, Cross AR, Hancock JT, Jones OT, Nakamura M, Kaever V, Resch K (1991) Functional expression of NADPH oxidase components (alpha- and beta-subunits of cytochrome b558 and 45-kDa flavoprotein) by intrinsic human glomerular mesangial cells. J Biol Chem 266(31):21025–21029
Segal AW, Harper AM, Cross AR, Jones OT (1986) Cytochrome b-245. Methods Enzymol 132:378–394
Cheng G, Cao Z, Xu X, Van Meir E, Lambeth J (2001) Homologs of gp91 phox: cloning and tissue expression of Nox3, Nox4, and Nox5. Gene 269:131–140
Paffenholz R, Bergstrom RA, Pasutto F, Wabnitz P, Munroe RJ, Jagla W, Heinzmann Y, Marquardt A, Bareiss A, Laufs J, Russ A, Stumm G, Schimenti JC, Bergstrom DE (2004) Vestibular defects in head-tilt mice result from mutations in nox3, encoding an NADPH oxidase. Genes Dev 18:486–491
Banfi B, Malgrange B, Knisz J, Steger K, Dubois-Dauphin M, Krause KH (2004) NOX3, a superoxide-generating NADPH oxidase of the inner ear. J Biol Chem 279:46065–46072
Nakano Y, Banfi B, Jesaitis AJ, Dinauer MC, Allen LA, Nauseef WM (2007) Critical roles for p22 phox in the structural maturation and subcellular targeting of Nox3. Biochem J 403(1):97–108
Aoyama T, Nagata K, Yamazoe Y, Kato R, Matsunaga E, Gelboin HV, Gonzalez FJ (1990) Cytochrome b 5 potentiation of cytochrome P-450 catalytic activity demonstrated by a vaccinia virus-mediated in situ reconstitution system. Proc Natl Acad Sci U S A 87:5425–5429
Borregaard N, Tauber AI (1984) Subcellular localization of the human neutrophil NADPH oxidase. b-cytochrome and associated flavoprotein. J Biol Chem 259:47–52
Nauseef WM (2014) Myeloperoxidase in human neutrophil host defence. Cell Microbiol 16(8):1146–1155. https://doi.org/10.1111/cmi.12312
Acknowledgments
The Nauseef lab is supported by National Institute of Health grants AI116546 and AI132335, a Merit Review award from the Veterans Affairs, and use of facilities at the Iowa City Department of Veterans Affairs Medical Center, Iowa City, IA.
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Nakano, Y., Nauseef, W.M. (2019). Spectroscopy of NOX Protein Family Members. In: Knaus, U., Leto, T. (eds) NADPH Oxidases. Methods in Molecular Biology, vol 1982. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9424-3_7
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DOI: https://doi.org/10.1007/978-1-4939-9424-3_7
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