Structure of human phagocyte cytochrome b and its relationship to microbicidal superoxide production

The human immune system depends on its phagocytes for on-demand production of toxic oxygen species at sites of infection, invasion, or injury. These species are formed because of the production of superoxide anion (O sub(2) super(-) times ) by the phagocyte nicotinamide-adenine dinucleotide phosphate (NADPH) super(2) oxidase. This multicomponent, activatable, membrane-enzyme complex transfers high energy electrons from cytoplasmic NADPH to molecular oxygen, permitting production of topologically extracellular O sub(2) super(-) times . At the functional center of this system is a unique integral membrane b-type cytochrome that functions as the redox "middleman" in this production. In the last 3 years, new structural and functional information about the oxidase has emerged that supports the functioning of that the molecule as an electron transferase for the reaction: NADPH + 2O sub(2) arrow right NADP super(+) + H super(+) + 2O sub(2) super(-) times . Human phagocyte cytochrome b, a.k.a. cytochrome b sub(559), cytochrome b sub(558), cytochrome b sub(-245), or flavocytochrome b, derives its name from spectral and functional properties. Room temperature-reduced minus oxidized absorbance spectra, originally used to classify and name cytochromes, identify it as a spectrally typical eukaryotic plasma membrane b-type cytochrome with a prominent alpha -absorbance band at 558 to 559 nm. At liquid nitrogen temperatures, the alpha -band splits into two, with peaks at 556 and 558 nm. The redox protein has a unique electrochemical mid point potential, originally determined to be -245 mV and more recently reflective of two heme centers with midpoint potentials of -225 and -265 mV super(3). The purified, reconstituted molecule also requires stoichiometric amounts of flavin-adenine dinucleotide (FAD) to function as a stand-alone vectorial superoxide-generating oxidase, supporting clues in its primary structure that suggest it may be a flavoprotein. The protein is composed of two polypeptides: a glycosylated heavy chain (gp91 super(phox) phagocyte oxidase component) M sub(r) = 91 kDa on SDS-PAGE, deglycosylated M sub(r) = 55-65 kDa) and a light, nonglycosylated polypeptide (p22 super(phox), M sub(r) = 22 kDa). These mass estimates were confirmed when heavy and light chain genes were cloned and sequenced. The predicted heavy and light chain polypeptide lengths (without coded N-terminal methionines) are 569 and 194 amino acid residues with calculated m.w. of 65,204 and 20,857