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Investigation into the structure of flavocytochrome b558

Wallach, Timothy Michael; (1998) Investigation into the structure of flavocytochrome b558. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Phagocytic cells play a key role in the immune system, engulfing and sequestering bacteria in a phagocytic vacuole. There they are subjected to a barrage of degradative enzymes and superoxide, O2-. Superoxide is generated by an electron transport chain called flavocytochrome b558, which spans the wall of the vacuole. The reactive products from superoxide, and the alteration in pH that these reactions cause, act together for successful bacterial killing. With its associated cytosolic factors, flavocytochrome b558 forms a complex called the NADPH Oxidase. If a component of the NADPH oxidase is missing or defective, so that superoxide is not produced, then the individual is severely immune-compromised, leading to the condition of Chronic Granulomatous Disease (CGD). Flavocytochrome is composed of two subunit types, p22phox and gp91phox. Together, these form an electron transport chain which uses cytosolic NADPH, via FAD and heme cofactors, to reduce molecular oxygen in the vacuolar space to superoxide. The subunits are closely associated and copurify through stringent conditions. There are now known to be two non-identical heme groups in the complex, and the ratio of heme to FAD is 2:1. The NADPH and FAD binding sites are on gp91phox, and the accepted view has been that heme was associated with p22phox. The work described in this thesis investigates the structure of flavocytochrome b558. First, the subunit stoichiometry was determined by a variety of complementary approaches. This data showed that p22phox and gp91phox were present in neutrophil membrane in a 1:1 ratio. Second, topology studies were carried out to analyze the glycosylation sites of gp91phox using site directed mutagenesis and in vitro protein synthesis. This information was used to construct a model of the amino-terminal, membrane associated part of gp91phox. The model is consistent with having both heme associated with gp91phox, along with the FAD and NADPH binding sites, implicating the p22phox subunit as having a structural and/or regulatory role in the oxidase.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Investigation into the structure of flavocytochrome b558
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Health and environmental sciences; Phagocytes
URI: https://discovery.ucl.ac.uk/id/eprint/10100772
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