Journal of Biological Chemistry
Volume 274, Issue 33, 13 August 1999, Pages 22999-23005
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ENZYMOLOGY
The p67phox Activation Domain Regulates Electron Flow from NADPH to Flavin in Flavocytochromeb 558*

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An activation domain in p67phox (residues within 199–210) is essential for cytochromeb 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 p67phox decreased the percent steady state reduction of the flavin to 4%, but omission of p47phox had little effect. The activation domain on p67phox was critical for regulating flavin reduction, since mutations in this region that decreased O⨪2generation also decreased the steady state reduction of flavin. Thus, the activation domain on p67phox 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 theR hydride transfer. The calculated K dfor NADPH was 40 μm in the presence of wild type p67phox and was ∼55 μm using the weakly activating p67phox(V205A). Thus, the activation domain of p67phox 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.

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This work was supported by National Institutes of Health Grant AI22809.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement ” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.