Hydrogen-deuterium exchange mass spectrometry for investigation of backbone dynamics of oxidized and reduced cytochrome P450cam
Hydrogen-deuterium exchange mass spectrometry for investigation of backbone dynamics of oxidized and reduced cytochrome P450cam
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DOI：
10.1016/j.jinorgbio.2007.10.001
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发表时间：
2008-02-01
影响因子：
3.9
通讯作者：
Pochapsky, Thomas C.
中科院分区：
文献类型：
--
作者：
Hamuro, Yoshitomo;Molnar, Kathleen S.;Pochapsky, Thomas C.
通过质谱法监测氢/氘交换（H/D交换），研究了樟脑单加氧酶细胞色素P450(cam) （CYP101）的主链动力学作为血红素铁氧化/连接状态的函数。主链酰胺NH氢可以很容易地与溶剂交换，这种交换的速率除其他外取决于局部结构元素的动态波动。多肽的流动区与溶剂的交换速度比受约束的区域更快。在酶的大多数区域，氧化的高自旋樟脑结合和还原的樟脑和co结合的CYP101（分别为cyps和cyps - co）之间的交换率相似。然而，在先前通过结构和分子动力学研究与底物接触有关的蛋白质区域，还原酶的交换速率明显低于氧化的cyps。这一观察结果对应于氧化酶相对于还原形式的灵活性增加。核磁共振测定的cyps - co在结合生物相关效应物和还原剂聚氧还蛋白（Pdx）时，先前发现的结构特征受到干扰，在还原状态下也更不易发生交换。据我们所知，这项研究代表了使用这种方法对P450家族的骨干动力学进行的首次实验研究。(c) 2007爱思唯尔公司版权所有。
Backbone dynamics of the camphor monoxygenase cytochrome P450(cam) (CYP101) as a function of oxidation/ligation state of the heme iron were investigated via hydrogen/deuterium exchange (H/D exchange) as monitored by mass spectrometry. Main chain amide NH hydrogens can exchange readily with solvent and the rate of this exchange depends upon, among other things, dynamic fluctuations in local structural elements. A fluxional region of the polypeptide will exchange more quickly with solvent than one that is more constrained. In most regions of the enzyme, exchange rates were similar between oxidized high-spin camphor-bound and reduced camphor- and CO-bound CYP101 (CYP-S and CYP-S-CO, respectively). However, in regions of the protein that have previously been implicated in substrate access by structural and molecular dynamics investigations, the reduced enzyme shows significantly slower exchange rates than the oxidized CYP-S. This observation corresponds to increased flexibility of the oxidized enzyme relative to the reduced form. Structural features previously found to be perturbed in CYP-S-CO upon binding of the biologically relevant effector and reductant putidaredoxin (Pdx) as determined by nuclear magnetic resonance are also more protected from exchange in the reduced state. To our knowledge, this study represents the first experimental investigation of backbone dynamics within the P450 family using this methodology. (c) 2007 Elsevier Inc. All rights reserved.