The cytosolic subunit p67 of the NADPH-oxidase complex does not bind NADPH

The NADPH-oxidase of phagocytic cells is a multicomponent enzyme that generates superoxide. It comprises a membrane flavocytochrome b₅₅₈ and four cytosolic proteins; p67^phox, p47^phox, p40^phox and Rac. The NADPH-binding site of this complex was shown to be located on the flavocytochrome b₅₅₈. However, a number of studies have suggested the presence of another site on the p67^phox subunit which is the key activating component. Using several approaches like tryptophan quenching fluorescence measurement, inhibition by 2′,3′-dialdehyde NADPH, and free/bound NADPH concentration measurements, we demonstrate that no NADPH binds on p67^phox, thus definitively solving the controversy on the number and location of the NADPH-binding sites on this complex.