Abstract
An activation domain in p67(phox) (residues within 199-210) is essential for cytochrome b(558)-dependent activation of NADPH superoxide (O2(-.)) 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 O2(-.) 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 microM in the presence of wild type p67(phox) and was approximately 55 microM 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.
Highlights
From the Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322 and the Department of Biochemistry, Aichi Medical University, Nagakute, Aichi 480-1195, Japan
The activation domain on p67phox was critical for regulating flavin reduction, since mutations in this region that decreased O2. generation decreased the steady state reduction of flavin
We recently identified an “activation domain” within p67phox that is essential for NADPH oxidase activity [36]
Summary
Materials—NADPH, NADPH analogs, FAD, arachidonic acid, cytochrome c (type IV, horse heart), thrombin, glutathione, n-octyl glucoside, cholic acid, GTP␥S, protease inhibitor mixture, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were purchased from Sigma. The resulting analog showed an absorption spectrum identical with that previously published for 8-nor-8-S-thioacetamido-FAD [37], and its concentration was determined using an extinction coefficient of 26 mMϪ1 cmϪ1 at 475 nm. The cell-free reaction mixtures included purified cytochrome b558 (275 nM) reconstituted with either native FAD or thioacetamido-FAD, 850 nM p47phox, 900 nM p67phox, 950 nM Rac preloaded with GTP␥S, and 200 –240 M arachidonate in a total volume of 50 l. Fluorescence changes at 525 nm induced by NADPH-FAD analog oxidoreduction during cell-free NADPH oxidase activation occurred slowly for about 5 min, and the total fluorescence change due to the complete reduction of the FAD analog was measured by adding a few crystals of sodium dithionite. The time course of heme reduction was derived from the absorbance changes at 558 minus 540 nm, using an extinction coefficient of 21.6 mMϪ1 cmϪ1 [48]
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