Functional interactions in cytochrome P450BM3. Fatty acid substrate binding alters electron-transfer properties of the flavoprotein domain
Functional interactions in cytochrome P450BM3. Fatty acid substrate binding alters electron-transfer properties of the flavoprotein domain
复制标题
DOI：
10.1021/bi961667u
复制
发表时间：
1996-11-26
期刊：
影响因子：
2.9
通讯作者：
Feyereisen, R
中科院分区：
文献类型：
--
作者：
Murataliev, MB;Feyereisen, R
P450BM3是一种细菌融合蛋白，位于细胞色素P450脂肪酸羟基酶(CyP102)和含有FAD和FMN的黄素蛋白之间，与NADPH：细胞色素P450还原酶同源。在没有脂肪酸底物的情况下，P450BM3与NADPH孵育会导致羟基酶活性的抑制[Narhi，L.O.，&Fulco，A.J.(1986)J.Biol]。化学。261、7160-7169]。我们发现，在这些条件下，月桂酸依赖的NADPH的氧化和氧气消耗也受到抑制。被抑制的酶不能将电子转移到血红素铁，但减少了人工电子受体，如细胞色素c、2，6-二氯苯酚或铁氰化铁。与这些受体孵育可迅速恢复P450BM3的羟基酶活性。该活性酶能同时催化细胞色素c还原和月桂酸羟化反应。细胞色素c对月桂酸羟化的K-m和V-max无影响。月桂酸盐和其他底物可刺激细胞色素c还原50%-70%。一氧化碳抑制羟基酶活性，但促进细胞色素c还原3-4倍，对细胞色素c的K-m没有影响。这种刺激需要在血红素催化部位结合底物。月桂酸结合诱导黄素蛋白结构域的构象变化，黄素荧光增加2倍。NADPH使P450BM3失活可消除月桂酸盐和CO对细胞色素c还原的刺激作用。羟基酶活性的完全抑制与细胞色素c还原的完全缺乏刺激有关。结果表明，在活性P450BM3中，这两个结构域的特定构象保持不变，从而确保了高羟基酶活性。P450BM3的细胞色素c还原酶和羟基酶活性涉及与Ravo蛋白结构域相互作用的不同部位、不同的催化中间体和不同的限速步骤。
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 accepters such as cytochrome c, 2,6-dichlorophenolindophenol, or ferricyanide. Incubation with these accepters 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 V-max 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 catalytic site. Laurate binding induces conformational changes in the flavoprotein domain as shown by a 2-fold increase of the flavin fluorescence. Inactivation of P450BM3 by NADPH abolishes the stimulation of cytochrome c reduction by laurate and CO. Complete inhibition of hydroxylase activity correlates with complete lack of stimulation of cytochrome c reduction. The results suggest that a specific conformation of the two domains is maintained in the active P450BM3, ensuring high hydroxylase activity. Cytochrome c reductase and hydroxylase activities of P450BM3 involve different sites of interaction with the Ravoprotein domain, different catalytic intermediates, and different rate-limiting steps.