Effector Roles of Putidaredoxin on Cytochrome P450cam Conformational States

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 (Fe2+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.

本研究通过将两种不同自旋标记物MTSL或BSL（双功能自旋标记物，bifunctional spin-label）连接至细胞色素P450cam（cytochrome P450cam）的F或G螺旋，并利用DEER（双电子-电子共振，double electron–electron resonance）测定标记物间的距离，对Pdx（普脱铁氧还蛋白，putidaredoxin）介导的细胞色素P450cam构象效应功能进行了精细化解析。此前的电子顺磁共振（EPR，electron paramagnetic resonance）与晶体学研究已观测到，氧化态Pdx可促使结合底物的P450cam从闭合构象转变为开放构象。然而，在结合一氧化碳的亚铁P450cam（Fe²⁺CO）与还原态Pdx形成的复合物中，DEER并未检测到该构象转变。此外，近期的核磁共振（NMR，nuclear magnetic resonance）研究同样未能观测到Pdx结合后P450cam的构象变化。因此，厘清上述矛盾结论，对于全面阐释Pdx的效应功能具有重要意义。本研究结果显示，当采用MTSL对F或G螺旋进行标记时，氧化态Pdx可诱导结合樟脑的P450cam从闭合构象向开放构象转变。在上述位点标记BSL，则可显著缩小距离分布的宽度，或改变构象转变的幅度。此外，本研究还报道了包含氧化态Pdx与结合一氧化碳的亚铁P450cam的混合氧化态样品的DEER谱图，结果显示P450cam仍维持闭合构象。这表明，血红素结合一氧化碳可阻断P450cam的构象开放，抵消Pdx结合所带来的调控影响。最后，本研究解析了结合底物的开放构象P450cam晶体结构，该结构提示晶体堆积效应可能阻碍了构象转换过程。通过多种标记策略，DEER为解析P450cam构象如何依赖于Pdx与配体状态提供了独特的研究视角。