Detection of a high-barrier conformational change in the active site of cytochrome P450cam upon binding of putidaredoxin
Detection of a high-barrier conformational change in the active site of cytochrome P450cam upon binding of putidaredoxin
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DOI：
10.1021/ja051195j
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发表时间：
2005-05-18
影响因子：
15
通讯作者：
Pochapsky, SS
中科院分区：
文献类型：
--
作者：
Wei, JY;Pochapsky, TC;Pochapsky, SS
用全氘代细胞色素P450 cam（CYP 101）、全氘代Pdx和同位素标记的樟脑，通过核磁共振研究了底物樟脑在还原CO结合的CYP 101活性部位的取向，并与还原putidaredoxin（Pdxr）的加成反应进行了比较。这使得CYP 101结合樟脑的1H共振被观察到，而不受CYP 101或Pdx信号的干扰，并确认结合形式的樟脑的甲基信号的归属。Cys 4Fe 2S 2铁氧还蛋白Pdx是CYP 101的生理氧化还原伴侣和效应子。将Pdx添加到还原的CYP 101 −樟脑−CO复合物中导致构象选择在化学位移时间尺度上缓慢，主要在樟脑的8-CH 3基团处观察到光谱效应。樟脑的信号是环电流移动的血红素，和9-和10-CH 3共振，这些位移是合理的预测从CO结合CYP 101的晶体结构的环电流计算。然而，在Pdx的情况下，CYP 101结合的樟脑的8-CH 3共振观察到在相当高的领域比预期的。动态模拟使用环电流位移限制产生的结构与低的化学位移违规，其中樟脑羰基氧和Tyr 96的OH之间的氢键丢失，和活性位点的膨胀发生，允许重新定位的樟脑内的活性位点。
The orientation of the substrate camphor in the active site of reduced CO-bound cytochrome P450cam(CYP101) as a function of reduced putidaredoxin (Pdxr) addition has been examined by NMR using perdeuterated CYP101 and perdeuterated Pdx as well as isotopically labeledd-camphor. This permits the1H resonances of CYP101-bound camphor to be observed without interference from the signals of CYP101 or Pdx and confirms assignments of the methyl signals of camphor in the bound form. The Cys4Fe2S2ferredoxin Pdx is the physiological redox partner and effector of CYP101. The addition of Pdx to the reduced CYP101−camphor−CO complex results in a conformational selection that is slow on the chemical shift time scale with spectral effects observed primarily at the 8-CH3group of the camphor. The camphor signals are ring current shifted by the heme, and for the 9- and 10-CH3resonances, these shifts are reasonably well predicted by ring current calculations from the crystal structure of CO-bound CYP101. However, in the absence of Pdx, the 8-CH3resonance of CYP101-bound camphor is observed at considerably higher field than predicted. Dynamic simulations using ring current shift restraints generated a structure with low chemical shift violations in which the hydrogen bond between the camphor carbonyl oxygen and the OH of Tyr96 is lost, and an expansion of the active site takes place that permits reorientation of the camphor within the active site.