Jenkins C M, Genzor C G, Fillat M F, Waterman M R, Gómez-Moreno C
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
J Biol Chem. 1997 Sep 5;272(36):22509-13. doi: 10.1074/jbc.272.36.22509.
Catalysis by microsomal cytochromes P450 requires the membrane-bound enzyme NADPH-cytochrome P450 reductase (P450 reductase), which transfers electrons to the P450 heme via a flavodoxin-like domain. Previously, we reported that Escherichia coli flavodoxin (Fld), a soluble electron transfer protein, directly interacts with bovine cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450c17) and donates electrons to this enzyme when reconstituted with NADPH-ferredoxin (flavodoxin) reductase (FNR) (Jenkins, C. M., and Waterman, M. R. (1994) J. Biol. Chem. 269, 27401-27408). To investigate whether flavodoxins can serve as useful models of the analogous domain in P450 reductase, we have examined the FNR-Fld system from the cyanobacterium Anabaena. Mutagenesis of two acidic Anabaena Fld residues (D144A and E145A) significantly decreased flavodoxin-supported P450c17 progesterone 17alpha-hydroxylase activity. Specifically, D144A exhibited only 15% of the activity of wild-type Fld, whereas the adjacent mutation, E145A, caused a 40% loss in activity. P450-dependent hydrogen peroxide/superoxide production by wild-type FNR-Fld was measurably higher than that generated by FNR-D144A or FNR-E145A, indicating that the mutations do not lead to P450 heme-mediated electron uncoupling. Interestingly, the D144A and E145A mutants bind with equal or even greater affinity to P450c17 than wild-type Fld. Furthermore, these mutations (D144A and E145A) actually increased cytochrome c reductase activity (35 and 100% higher than wild type). Anabaena Fld residues Asp144 and Glu145 align closely with rat P450 reductase residue Asp208, which has been shown by mutagenesis to be important in electron transfer to P4502B1 but not to cytochrome c (Shen, A. L., and Kasper, C. B. (1995) J. Biol. Chem. 270, 27475-27480). Thus, these residues in flavodoxins and P450 reductase appear to have similar functions in P450 recognition and/or electron transfer, supporting the hypothesis that flavodoxins represent valid models for the FMN-binding domain of P450 reductase.
微粒体细胞色素P450的催化作用需要膜结合酶NADPH - 细胞色素P450还原酶（P450还原酶），该酶通过类黄素氧还蛋白结构域将电子转移到P450血红素上。此前，我们报道过，大肠杆菌黄素氧还蛋白（Fld），一种可溶性电子转移蛋白，可直接与牛细胞色素P450 17α - 羟化酶/17,20 - 裂解酶（P450c17）相互作用，并在与NADPH - 铁氧还蛋白（黄素氧还蛋白）还原酶（FNR）重组时将电子传递给该酶（詹金斯，C.M.，和沃特曼，M.R.（1994年）《生物化学杂志》269卷，27401 - 27408页）。为了研究黄素氧还蛋白是否可作为P450还原酶中类似结构域的有用模型，我们研究了蓝藻鱼腥藻的FNR - Fld系统。对鱼腥藻Fld的两个酸性残基（D144A和E145A）进行诱变显著降低了黄素氧还蛋白支持的P450c17孕酮17α - 羟化酶活性。具体而言，D144A仅表现出野生型Fld活性的15%，而相邻突变E145A导致活性损失40%。野生型FNR - Fld依赖P450产生的过氧化氢/超氧化物的量明显高于FNR - D144A或FNR - E145A产生的量，这表明这些突变不会导致P450血红素介导的电子解偶联。有趣的是，D144A和E145A突变体与P450c17的结合亲和力与野生型Fld相当甚至更高。此外，这些突变（D144A和E145A）实际上增加了细胞色素c还原酶活性（比野生型高35%和100%）。鱼腥藻Fld的Asp144和Glu145残基与大鼠P450还原酶的Asp208残基紧密对齐，诱变已表明该残基在向P4502B1传递电子方面很重要，但对细胞色素c则不然（沈，A.L.，和卡斯珀，C.B.（1995年）《生物化学杂志》270卷，27475 - 27480页）。因此，黄素氧还蛋白和P450还原酶中的这些残基在P450识别和/或电子转移方面似乎具有相似功能，支持了黄素氧还蛋白代表P450还原酶FMN结合结构域有效模型的假说。
