Assembly of the neutrophil respiratory burst oxidase: A direct interaction between p67^PHOX and cytochrome b₅₅₈

Abstract

Activation of the phagocyte NADPH oxidase complex requires the assembly of the cytosolic factors p47^PHOX, p67^PHOX, p40^PHOX, and Rac1 or Rac2, with the membrane-bound cytochrome b ₅₅₈. Whereas the interaction of p47^PHOX with cytochrome b ₅₅₈ is well established, an interaction between p67^PHOX and cytochrome b ₅₅₈ has never been investigated. We report here a direct interaction between p67^PHOX and cytochrome b ₅₅₈. First, labeled p67^PHOX recognizes a 91-kDa band in specific granules from a normal patient but not from a cytochrome b ₅₅₈-deficient patient. Second, p67^PHOX binds to cytochrome b ₅₅₈ that has been bound to nitrocellulose. Third, GTP-p67^PHOX bound to glutathione agarose is able to pull down cytochrome b _558. Rac1-GTP or Rac1-GDP increased the binding of p67^PHOX to cytochrome b ₅₅₈, suggesting that at least one of the oxidase-related functions of Rac1 is to promote the interaction between p67^PHOX and cytochrome b ₅₅₈.