Department of Chemistry, University of California, Davis , One Shields Avenue, Davis, California 95616, United States.
J Am Chem Soc. 2016 Aug 17;138(32):10163-72. doi: 10.1021/jacs.6b04110. Epub 2016 Aug 5.
In this study, the effector role of Pdx (putidaredoxin) on cytochrome P450cam conformation is refined by attaching two different spin labels, MTSL or BSL (bifunctional spin-label) onto the F or G helices and using DEER (double electron-electron resonance) to measure the distance between labels. Recent EPR and crystallographic studies have observed that oxidized Pdx induces substrate-bound P450cam to change from the closed to the open state. However, this change was not observed by DEER in the reduced Pdx complex with carbon-monoxide-bound P450cam (Fe(2+)CO). In addition, recent NMR studies have failed to observe a change in P450cam conformation upon binding Pdx. Hence, resolving these issues is important for a full understanding the effector role of Pdx. Here we show that oxidized Pdx induces camphor-bound P450cam to shift from the closed to the open conformation when labeled on either the F or G helices with MTSL. BSL at these sites can either narrow the distance distribution widths dramatically or alter the extent of the conformational change. In addition, we report DEER spectra on a mixed oxidation state containing oxidized Pdx and ferrous CO-bound P450cam, showing that P450cam remains closed. This indicates that CO binding to the heme prevents P450cam from opening, overriding the influence exerted by Pdx binding. Finally, we report the open form P450cam crystal structure with substrate bound, which suggests that crystal packing effects may prevent conformational conversion. Using multiple labeling approaches, DEER provides a unique perspective to resolve how the conformation of P450cam depends on Pdx and ligand states.
在这项研究中，通过将两个不同的自旋标记物，MTSL 或 BSL（双功能自旋标记物）连接到 F 或 G 螺旋上，并使用 DEER（双电子电子共振）测量标记物之间的距离，来细化 Pdx（putidaredoxin）对细胞色素 P450cam 构象的效应器作用。最近的 EPR 和晶体学研究表明，氧化态 Pdx 诱导底物结合的 P450cam 从闭合状态转变为开放状态。然而，在还原态 Pdx 与一氧化碳结合的 P450cam（Fe（2+）CO）复合物中，DEER 并未观察到这种变化。此外，最近的 NMR 研究未能观察到 Pdx 结合时 P450cam 构象的变化。因此，解决这些问题对于全面理解 Pdx 的效应器作用至关重要。在这里，我们表明，当在 F 或 G 螺旋上用 MTSL 标记时，氧化态 Pdx 诱导樟脑结合的 P450cam 从闭合状态转变为开放状态。在这些位点的 BSL 可以显著缩小距离分布宽度或改变构象变化的程度。此外，我们报告了含有氧化态 Pdx 和亚铁一氧化碳结合的 P450cam 的混合氧化态的 DEER 光谱，表明 P450cam 仍然处于闭合状态。这表明一氧化碳与血红素的结合阻止了 P450cam 的打开，这超过了 Pdx 结合所施加的影响。最后，我们报告了结合有底物的开放形式 P450cam 晶体结构，这表明晶体堆积效应可能会阻止构象转换。使用多种标记方法，DEER 提供了一种独特的视角来解决 P450cam 的构象如何取决于 Pdx 和配体状态。
