Functional interactions in cytochrome P450BM3. Fatty acid substrate binding alters electron-transfer properties of the flavoprotein domain

Biochemistry
M B Murataliev, R Feyereisen

Abstract

P450BM3 is a bacterial fusion protein between a cytochrome P450 fatty acid hydroxylase (CYP102) and an FAD- and FMN-containing flavoprotein homologous to NADPH: cytochrome P450 reductase. It has been shown that incubation of P450BM3 with NADPH in the absence of a fatty acid substrate results in inhibition of hydroxylase activity [Narhi, L. O., & Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169]. We show that laurate-dependent oxidation of NADPH and oxygen consumption are also inhibited under those conditions. The inhibited enzyme is unable to transfer electrons to the heme iron, but reduces artificial electron acceptors such as cytochrome c, 2,6-dichlorophenolindophenol, or ferricyanide. Incubation with these acceptors rapidly restores hydroxylase activity of P450BM3. The active enzyme is able to catalyze the reduction of cytochrome c and hydroxylation of laurate simultaneously. Cytochrome c has no effect on the K(m) and Vmax of laurate hydroxylation. Laurate and other substrates stimulate cytochrome c reduction by 50-70%. Carbon monoxide inhibits hydroxylase activity, but stimulates cytochrome c reduction 3-4 fold and has no effect on the K(m) for cytochrome c. This stimulation requires binding of a substrate at the heme cata...Continue Reading

Citations

Apr 6, 2004·Biochimica Et Biophysica Acta·Marat B MuratalievF Ann Walker
Sep 19, 2002·Journal of Inorganic Biochemistry·Shigeyoshu FuziwaraToru Shimizu
Dec 4, 2012·International Journal of Molecular Sciences·Louise AigrainGilles Truan
May 23, 2001·Archives of Biochemistry and Biophysics·L A CowartJ H Capdevila
Apr 9, 2008·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Chiung-Kuang J ChenF Ann Walker
Sep 4, 2009·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Chiung-Kuang J ChenF Ann Walker
Jul 16, 2008·Journal of Molecular Biology·Rudi FasanFrances H Arnold
Oct 7, 2006·Biochimica Et Biophysica Acta·Andrew W MunroKirsty J McLean
Nov 11, 1998·Archives of Biochemistry and Biophysics·V M GuzovR Feyereisen
Oct 20, 2011·Chemical Society Reviews·Christopher J C WhitehouseLuet-Lok Wong
Dec 26, 2017·Chembiochem : a European Journal of Chemical Biology·Gloria Saab-RincónRudi Fasan
May 1, 2007·Journal of the American Chemical Society·Hazel M GirvanAndrew W Munro

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