Chudaev M V, Gilep A A, Usanov S A
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, 220141, Belarus.
Biochemistry (Mosc). 2001 Jun;66(6):667-81. doi: 10.1023/a:1010215516226.
We have shown earlier that microsomal cytochrome b5 can form a specific complex with mitochondrial cytochrome P450 (cytochrome P450scc). The formation of the complex between these two heme proteins was proved spectrophotometrically, by affinity chromatography on immobilized cytochrome b5, and by measuring the cholesterol side-chain cleavage activity of cytochrome P450scc in a reconstituted system in the presence of cytochrome b5. To further study the mechanism of interaction of these heme proteins and evaluate the role of negatively charged amino acid residues Glu42, Glu48, and Asp65 of cytochrome b5, which are located at the site responsible for interaction with electron transfer partners, we used site-directed mutagenesis to replace residues Glu42 and Glu48 with lysine and residue Asp65 with alanine. The resulting mutant forms of cytochrome b5 were expressed in E. coli, and full-length and truncated forms (shortened from the C-terminal sequence due to cleavage of 40 amino acid residues) of these cytochrome b5 mutants were purified. Addition of the truncated forms of cytochrome b5 (which do not contain the hydrophobic C-terminal sequence responsible for interaction with the membrane) to the reconstituted system containing cytochrome P450scc caused practically no stimulation of catalytic activity, indicating an important role of the hydrophobic fragment of cytochrome b5 in its interaction with cytochrome P450scc. However, full-length cytochrome b5 and the full-length Glu48Lys and Asp65Ala mutant forms of cytochrome b5 stimulated the cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc by 100%, suggesting that residues Glu48 and Asp65 of cytochrome b5 are not directly involved in its interaction with cytochrome P450scc. The replacement of Glu42 for lysine, however, made the Glu42Lys mutant form of cytochrome b5 about 40% less effective in stimulation of the cholesterol side-chain cleavage activity of cytochrome P450scc, indicating that residue Glu42 of cytochrome b5 is involved in electrostatic interactions with cytochrome P450scc. Residues Glu42 and Glu48 of cytochrome b5 appear to participate in electrostatic interaction with microsomal type cytochrome P450.
我们之前已经表明，微粒体细胞色素b5可与线粒体细胞色素P450（细胞色素P450scc）形成特定复合物。通过分光光度法、固定化细胞色素b5的亲和层析以及在细胞色素b5存在的重构系统中测量细胞色素P450scc的胆固醇侧链裂解活性，证实了这两种血红素蛋白之间复合物的形成。为了进一步研究这些血红素蛋白的相互作用机制，并评估细胞色素b5中位于与电子传递伙伴相互作用位点的带负电荷氨基酸残基Glu42、Glu48和Asp65的作用，我们使用定点诱变将残基Glu42和Glu48替换为赖氨酸，将残基Asp65替换为丙氨酸。所得细胞色素b5的突变形式在大肠杆菌中表达，并对这些细胞色素b5突变体的全长和截短形式（由于40个氨基酸残基的切割而从C端序列缩短）进行了纯化。将细胞色素b5的截短形式（不包含负责与膜相互作用的疏水C端序列）添加到含有细胞色素P450scc的重构系统中，几乎不会刺激催化活性，这表明细胞色素b5的疏水片段在其与细胞色素P450scc的相互作用中起重要作用。然而，全长细胞色素b5以及细胞色素b5的全长Glu48Lys和Asp65Ala突变形式将细胞色素P450scc催化的胆固醇侧链裂解反应刺激了100%，这表明细胞色素b5的残基Glu48和Asp65不直接参与其与细胞色素P450scc的相互作用。然而，将Glu42替换为赖氨酸使细胞色素b5的Glu42Lys突变形式在刺激细胞色素P450scc的胆固醇侧链裂解活性方面的效果降低了约40%，这表明细胞色素b5的残基Glu42参与了与细胞色素P450scc的静电相互作用。细胞色素b5的残基Glu42和Glu48似乎参与了与微粒体类型细胞色素P450的静电相互作用。
