NDLI: The p67 phox Activation Domain Regulates Electron Flow from NADPH to Flavin in Flavocytochromeb 558

Content Provider	Journal of Biological Chemistry (JBC)
Author	Nisimoto, Yukio Motalebi, Shabnam Han, Chang-Hoon Lambeth, J. David
Abstract	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.
Related Links	http://www.jbc.org/content/274/33/22999.abstract
Ending Page	23005
Starting Page	22999
Page Count	7
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	33
Volume Number	274
DOI	10.1074/jbc.274.33.22999
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	1999-08-13
Access Restriction	Open
Subject Keyword	Superoxide anion (O⨪2) Guanosine 5′-(γ-thio)triphosphate (GTPγS) L-α-phosphatidylcholine (PC) L-α-phosphatidylethanolamine (PE) L-α-phosphatidylinositol (PI) Sphingomyelin (SM) Monodeuterated form of NADPH (NADPD) ENZYMOLOGY
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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