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   Cytochrome b558 is comprised of two subunits, gp91phox and p22phox. The large subunit actually encodes a protein of about 65 kDa, but glycosylation causes it to run as a diffuse band around 91 kDa on SDS gels. gp91phox contains a region of homology with the NADPH-binding region of several flavoenzymes including cytochrome P450 reductase and ferredoxin-NADP+ reductase, and also show weaker homology with the FAD binding site of several flavoprotein dehydrogenases. The figure also shows point mutations in variants of Chronic Granulomatous Disease which inhibit NADPH oxidase activity as well as proposed mechanisms in some cases.

   In addition to the pyridine nucleotide binding site homology sequence, data from ours and from other laboratories indicate that the cytochrome binds FAD and its analogs, and contains two hemes for each FAD. Early studies missed the presence of FAD due to the inability to recover FAD after detergent extraction, but the flavin can be reconstituted in the presence of phospholipids. Thus, the cytochrome contains all of the known redox centers of the respiratory burst oxidase. A model of the large subunit can be constructed based on a combination of hydropathy analysis and available information regarding homology of N-terminal regions with heme binding regions of known hemoproteins as well as homology with known flavoprotein dehydrogenases. According to this model, the very hydrophobic N terminal 225 or so amino acids loops back and forth across the plasma membrane at least 5 times. This region also contains the two hemes (light red structures), which we suggest lie in a transmembrane arrangement. The C-terminal two thirds of the molecule contains the homology regions for NADPH and FAD binding, and is proposed to fold into a globular domain analogous to known flavoprotein dehydrogenases.

   The small subunit, p22phox, contains a two proline-rich sequences at the C-terminus, one of which is similar to the consensus sequence derived by Baltimore's lab for binding to SH3 regions. This domain has been demonstrated to bind to one of the SH3 domains of p47phox. In a structural model, the sequence also contains a hydrophobic N-terminus which can be predicted to loop back and forth across the membrane probably three times. The small subunit has a single histidine which is conserved across species, and this residue has been proposed to cooperate with the large subunit to form one of the heme binding sites. This arrangement places the proline-rich C-terminus on the cytosolic side of the membrane where it can interact with p47phox.

   A central question in the NADPH oxidase field remains how does complexation with the cytosolic factors regulate the catalytic activity of the flaovocytochrome.

More Reading:

Takeshige, K. and Sumimoto, H. (1994) "Cytochrome b558: A Flavocytochrome comprising the complete electron-transporting apparatus of phagocyte NADPH oxidase" in Regulation of Heme Protein Synthesis 1994, pp. 97-102.

Finegold, A.A., Shatwell, K.P., Segal, A.W., Klausner, R.D. and Dancis, A. (1996) "Intramembrane Bis-Heme Motif for Transmembrane Electron Transport Conseved in Yeast Iron Reductase and the Human NADPH Oxidase" J. Biol. Chem. 271, 31021-31024.

Nisimoto, Y., Otsuka-Murakami, H.and Lambeth, J.D. (1995) "Reconstitution of flavin-depleted neutrophil flavocytochrome b558 with 8-mercapto-FAD and characterization of the flavin-reconstituted enzyme" J. Biol. Chem. 270, 16428-16434.

Uhlinger, D.J., Tyagi, S.R. and Lambeth, J.D. (1995) "On the mechanism of inhibition of the neutrophil respiratory burst oxidase by a peptide from the C-terminus of the large subunit of cytochrome b558" Biochemistry 34, 524-527.

Tamura, M., Tamura, T., Kugi, M., Takeshita,M. and Lambeth, J.D. (1992) "Stabilization of Human Neutrophil NADPH-Oxidase Activated in a Cell-Free System by Cross-Linking", J. Biol. Chem. 266, 7529-7538.

Taylor, W.R., Jones, D.T. and Segal, A.W. (1993) "A Structural Model for the Nucleotide Binding Domains of the Flavocytochrome b-245 ß chain" Protein Science 2, 1675-1685.