The p67phox activation domain regulates electron flow from NADPH to flavin in flavocytochrome b558

An activation domain in p67(phox) (residues within 199-210) is essential for cytochrome b(558)-dependent activation of NADPH superoxide (O-2(.-)) generation in a cell-free system (Han, C.-H., Freeman, J. L. R., Lee, T., Motalebi, S. A., and Lambeth, J. D. (1998) J. Biol. Chem. 273, 16663-16668). To determine the steady state reduction flavin in the presence of highly absorbing hemes, 8-nor-8-S-thioacetamido-FAD ("thioacetamido-FAD") was reconstituted into the flavocytochrome, and the fluorescence of its oxidized form was monitored. Thioacetamido-FAD-reconstituted cytochrome showed lower activity (7% versus 100%) and increased steady state flavin reduction (28 versus <5%) compared with the enzyme reconstituted with native FAD, Omission of p67(phox) decreased the percent steady state reduction of the flavin to 4%, but omission of p47(phox) had little effect. The activation domain on p67(phox) was critical for regulating flavin reduction, since mutations in this region that decreased O-2(.-) generation also decreased the steady state reduction of flavin, Thus, the activation domain on p67(phox) regulates the reductive half-reaction for FAD, This reaction is comprised of the binding of NADPH followed by hydride transfer to the flavin, Kinetic deuterium isotope effects along with K-m values permitted calculation of the K-d for NADPH, (R)-NADPD but not (S)-NADPD showed kinetic deuterium isotope effects on V and V/K of about 1.9 and 1.5, respectively, demonstrating stereospecificity for the R hydride transfer. The calculated K-d for NADPH was 40 mu M in the presence of wild type p67(phox) and was similar to 55 mu M using the weakly activating p67(phox)(V205A), Thus, the activation domain of p67(phox) regulates the reduction of FAD but has only a small effect on NADPH binding, consistent with a dominant effect on hydride/electron transfer from NADPH to FAD.