Heme-ligating histidines in flavocytochrome b₅₅₈: Identification of specific histidines in gp91^phox

The phagocyte NADPH-dependent oxidase generates superoxide (O ₂^.-) by reducing molecular oxygen through flavocytochrome b₅₅₈ (flavocytochrome b), a heterodimeric oxidoreductase composed of gp91^phox and p22^phox subunits. Although each flavocytochrome b molecule contains two heme groups, their precise distribution within the heterodimer is unknown. Among functionally and/or structurally related oxidoreductases, histidines at codons 101, 111, 115, 119, 209, 210, and 222 of gp91^phox are conserved and potential candidates to ligate heme. We compared biochemical and functional features of normal flavocytochrome b with those in cells expressing gp91^phox harboring amino acid substitutions at each of these histidines. Surface expression of flavocytochrome b and heterodimer formation were relatively unaffected in cells expressing gp91^phox H111L, H119L, or H210L. These mutations also had no effect on the flavocytochrome b heme spectrum, although NADPH oxidase activity was decreased in cells expressing gp91^phox H119L or H210L. In contrast, gp65 was not processed to gp91^phox, heterodimers did not form, and flavocytochrome b was not expressed on the surface of cells expressing gp91^phox H101L, H115L, H115D, H209C, H209Y, H222L, H222C, or H222R. Similarly, this subset of mutants lacked detectable O₂ ^.--generating activity, and flavocytochrome b purified from these cells contained little or no heme. These findings demonstrate that His ¹⁰¹, His¹¹⁵, His²⁰⁹, and His²²² of gp91^phox are critical for heme binding and biosynthetic maturation of flavocytochrome b.