Characterization of surface structure and p41^phox SH3 domain-mediated conformational changes for human neutrophil flavocytochrome b

The heterodimeric, integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the phagocyte NADPH oxidase and generates superoxide which plays a critical role in host defense. To better define the activation of superoxide production by this multisubunit enzyme complex, Cyt b-specific monoclonal antibodies (mAbs) and the p41^phox SH3 domains (P47SH3 _AB) were used in the present study as probes to map surface structure and conformational dynamics in human neutrophil Cyt b. In pull-down and co-immunoprecipitation studies with detergent-solubilized Cyt b, the oxidase-inhibitory mAb CS9 was shown to share an overlapping binding site with P47SH3_AB on the C-terminal region of the p22^phox subunit. Similar studies demonstrated a surprising lack of overlap between the mAb 44.1 and CS9/P47SH3_AB binding sites, and they indicated that the oxidase-inhibitory mAb NL7 binds a region physically separated from the p22 ^phox C-terminal domain. Resonance energy transfer and size exclusion chromatography confirmed the above results for functionally reconstituted Cyt b and provided evidence that binding of both mAb CS9 and P47SH3_AB altered the conformation of Cyt b. Further support that binding of the p47 ^phox SH3 domains modulates the structure of Cyt b was obtained using a cell-free assay system where P47SH3_AB enhanced superoxide production in the presence of a p67^phox (1 -212)-Rac1(Q61L) fusion protein. Taken together, this study further characterizes the structure of human neutrophil Cyt b in both detergent micelles and reconstituted membrane bilayers, and it provides evidence that the cytosolic regulatory subunit p47^phox modulates the conformation of Cyt b (in addition to serving as an adapter protein) during oxidase activation.