Spatial and electrogenic properties of superoxide-producing cytochrome b-559 incorporated into liposomes.
Purified cytochrome b-559 reconstituted into liposomes, consisting of certain azolectin-based phospholipid mixtures, is capable of NADPH-supported FAD-dependent superoxide (O2-) production in the absence of cytosolic activators. This system, representing the simplest model of the respiratory burst NADPH oxidase, was used to study cytochrome b-559 enzymology and distinguish putative mechanisms of NADPH oxidase activation (Koshkin, V. and Pick, E. (1993) FEBS Lett. 327, 57-62; (1994) FEBS Lett. 338, 285-289). In the present report, representing an extension of our earlier investigations, two types of vesicle-incorporated and reflavinated cytochrome b-559 preparation were distinguished by their ability to catalyze vectorial electrogenic or scalar electron transport from NADPH to oxygen. This can be explained by the existence of two distinct membranal locations of cytochrome b-559, with NADPH-binding and O2-reducing sites exposed on different or on the same side of the membrane. The mode of cytochrome b-559 insertion into the membrane depended on the reconstitution method employed. Both states of the reconstituted cytochrome b-559 were functionally competent judging by their susceptibility to additional activation by cytosolic NADPH oxidase components. The capability of flavocytochrome b-559 to function as a transmembrane electrogenic electron carrier points to its crucial role in the respiratory burst not only in its catalytical but also in its vectorial aspect. The scalar mode of its action may be related to respiratory burst pathology.
Koshkin
V
V
Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Israel.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003573:Cytochrome b Group / Q000737:chemistry* / Q000302:isolation & purification; D004579:Electron Transport; D006168:Guinea Pigs; D008081:Liposomes; D017737:Macrophages, Peritoneal / Q000201:enzymology; D009249:NADP / Q000737:chemistry; D045332:Photosystem II Protein Complex; D013481:Superoxides / Q000737:chemistry*
D003573:Cytochrome b Group; D008081:Liposomes; D045332:Photosystem II Protein Complex; D013481:Superoxides; D009249:NADP; C019313:cytochrome b559