Raman evidence for specific substrate-induced structural changes in the heme pocket of human cytochrome P450 aromatase during the three consecutive oxygen activation steps.
Raman evidence for specific substrate-induced structural changes in the heme pocket of human cytochrome P450 aromatase during the three consecutive oxygen activation steps.
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在三个连续的氧活化步骤中，人细胞色素 P450 芳香酶血红素口袋中特定底物诱导的结构变化的拉曼证据。
DOI：
10.1021/bi060094a
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发表时间：
2006
期刊：
影响因子：
2.9
通讯作者：
Kitagawa,Teizo
中科院分区：
文献类型：
--
作者：
Tosha,Takehiko;Kagawa,Norio;Ohta,Takehiro;Yoshioka,Shiro;Waterman,MichaelR;Kitagawa,Teizo
提出了人细胞色素 P450 芳香酶 (P450arom) 血红素口袋中特定底物诱导的结构变化，该酶经历了三个连续的氧活化步骤。我们通过纯化酶的无底物和底物结合形式的共振拉曼光谱对这种血红素环境进行了实验研究。 CO复合物的Fe−CO拉伸模式（νFe-CO）和氧化形式的Fe3+−S拉伸模式（νFe-S）分别作为血红素远端和近端侧的结构标记进行监测。通过分别作为第一步和第三步的底物的雄烯二酮和 19-醛-雄烯二酮的结合，νFe-CO 模式从 477 升高到 485 和 490 cm-1，而 P450arom 与 19-羟基雄烯二酮（第二步的底物）的结合没有观察到 νFe-CO，这表明血红素远端位点非常灵活，并且根据情况改变其结构。在基材上。 19-醛-雄烯二酮结合可以减少轴向硫醇盐的电子供给，与雄烯二酮结合的 P450arom 相比，νFe-Sby 5 cm-1 的低频偏移可以明显看出这一点。血红素远端位点环境的变化以及 19-醛-雄烯二酮结合时轴向硫醇盐的电子供给减少可能会稳定过氧化铁物质（第三步的活性中间体），同时抑制第一步和第二步的活性物质化合物 I (Fe4+O 卟啉+·) 的形成。因此，我们建议底物可以通过调节 P450arom 血红素远端和近端位点的结构来调节替代反应中间体的形成。
Specific substrate-induced structural changes in the heme pocket are proposed for human cytochrome P450 aromatase (P450arom) which undergoes three consecutive oxygen activation steps. We have experimentally investigated this heme environment by resonance Raman spectra of both substrate-free and substrate-bound forms of the purified enzyme. The Fe−CO stretching mode (νFe-CO) of the CO complex and Fe3+−S stretching mode (νFe-S) of the oxidized form were monitored as a structural marker of the distal and proximal sides of the heme, respectively. The νFe-COmode was upshifted from 477 to 485 and to 490 cm-1by the binding of androstenedione and 19-aldehyde-androstenedione, substrates for the first and third steps, respectively, whereas νFe-COwas not observed for P450arom with 19-hydroxyandrostenedione, a substrate for the second step, indicating that the heme distal site is very flexible and changes its structure depending on the substrate. The 19-aldehyde-androstenedione binding could reduce the electron donation from the axial thiolate, which was evident from the low-frequency shift of νFe-Sby 5 cm-1compared to that of androstenedione-bound P450arom. Changes in the environment in the heme distal site and the reduced electron donation from the axial thiolate upon 19-aldehyde-androstenedione binding might stabilize the ferric peroxo species, an active intermediate for the third step, with the suppression of the formation of compound I (Fe4+O porphyrin+•) that is the active species for the first and second steps. We, therefore, propose that the substrates can regulate the formation of alternative reaction intermediates by modulating the structure on both the heme distal and proximal sites in P450arom.