Tosha Takehiko, Kagawa Norio, Ohta Takehiro, Yoshioka Shiro, Waterman Michael R, Kitagawa Teizo
Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki 444-8787, Japan.
Biochemistry. 2006 May 2;45(17):5631-40. doi: 10.1021/bi060094a.
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 (nu(Fe)(-)(CO)) of the CO complex and Fe(3+)-S stretching mode (nu(Fe)(-)(S)) of the oxidized form were monitored as a structural marker of the distal and proximal sides of the heme, respectively. The nu(Fe)(-)(CO) mode was upshifted from 477 to 485 and to 490 cm(-)(1) by the binding of androstenedione and 19-aldehyde-androstenedione, substrates for the first and third steps, respectively, whereas nu(Fe)(-)(CO) was 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 nu(Fe)(-)(S) by 5 cm(-)(1) compared 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 (Fe(4+)=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.
有人提出，参与连续三步氧激活过程的人细胞色素P450芳香化酶（P450arom）的血红素口袋会发生特定底物诱导的结构变化。我们通过对纯化酶的无底物和底物结合形式进行共振拉曼光谱实验研究了这种血红素环境。分别监测了CO复合物的Fe-CO伸缩模式（ν(Fe)(-)(CO)）和氧化形式的Fe(3+)-S伸缩模式（ν(Fe)(-)(S)），作为血红素远端和近端的结构标记。雄烯二酮和19-醛-雄烯二酮（分别为第一步和第三步的底物）的结合使ν(Fe)(-)(CO)模式从477 cm(-)(1)分别上移至485 cm(-)(1)和490 cm(-)(1)，而对于第二步底物19-羟基雄烯二酮的P450arom，未观察到ν(Fe)(-)(CO)，这表明血红素远端位点非常灵活，其结构会根据底物而变化。19-醛-雄烯二酮的结合会减少轴向硫醇盐的电子供体作用，与结合雄烯二酮的P450arom相比，ν(Fe)(-)(S)的低频位移5 cm(-)(1)就证明了这一点。19-醛-雄烯二酮结合后血红素远端位点环境的变化以及轴向硫醇盐电子供体作用的减少可能会稳定铁过氧物种（第三步的活性中间体），同时抑制化合物I（Fe(4+)=O卟啉(+)(*)）的形成，而化合物I是第一步和第二步的活性物种。因此，我们提出底物可以通过调节P450arom中血红素远端和近端位点的结构来调控替代反应中间体的形成。
