Heme-ligating histidines in flavocytochrome b(558): identification of specific histidines in gp91(phox).
The phagocyte NADPH-dependent oxidase generates superoxide (O(2)) by reducing molecular oxygen through flavocytochrome b(558) (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 O(2)-generating activity, and flavocytochrome b purified from these cells contained little or no heme. These findings demonstrate that His(101), His(115), His(209), and His(222) of gp91(phox) are critical for heme binding and biosynthetic maturation of flavocytochrome b.
Biberstine-Kinkade
K J
KJ
Department of Pediatrics (Hematology/Oncology), Wells Center for Pediatric Research, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
DeLeo
F R
FR
Epstein
R I
RI
LeRoy
B A
BA
Nauseef
W M
WM
Dinauer
M C
MC
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D003573:Cytochrome b Group / Q000096:biosynthesis / Q000737:chemistry*; D019281:Dimerization; D006418:Heme / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D013329:Structure-Activity Relationship
D003573:Cytochrome b Group; D008562:Membrane Glycoproteins; D006418:Heme; D006639:Histidine; C036888:cytochrome b558; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases