Dynamics underlying hydroxylation selectivity of cytochrome P450cam
Dynamics underlying hydroxylation selectivity of cytochrome P450cam
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DOI：
10.1016/j.bpj.2021.01.027
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发表时间：
2021-03-02
影响因子：
3.4
通讯作者：
Thielges，Megan C.
中科院分区：
文献类型：
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作者：
Ramos，Sashary;Mammoser，Claire C.;Thielges，Megan C.
酶与底物的复合物的结构异质性和动力学可以决定催化的选择性;然而，充分表征如何仍然具有挑战性，因为异质性和动力学可以在氨基酸残基的空间水平上变化，并且涉及快速的时间尺度。我们展示了新生的方法，特定于网站的二维红外（IR）光谱研究的原型细胞色素P450，P450 CAM，以更好地描绘的机制较低的区域选择性的羟基化的底物降樟脑相比，本机底物樟脑。通过选择性地引入在蛋白质IR光谱的光谱隔离区域中具有频率的氰基作为局部振动探针，在整个酶中靶向特定位置。线性和二维红外光谱被施加到测量在每个探针的异质性和动力学，并探讨他们如何区分樟脑和降樟脑识别。红外光谱数据表明，去甲樟脑复合物并没有完全诱导大规模的构象变化，在樟脑复合物中采用的酶的封闭状态。此外，针对绑定基板的探针经验迅速相互转换的国家在去甲樟脑复杂的解释羟基化产物分布。总而言之，这项研究揭示了大规模和小规模的结构异质性和动力学，可能有助于细胞色素P450的选择性，并说明了位点选择性红外光谱法来阐明蛋白质动力学的方法。
Structural heterogeneity and the dynamics of the complexes of enzymes with substrates can determine the selectivity of catalysis; however, fully characterizing how remains challenging as heterogeneity and dynamics can vary at the spatial level of an amino acid residue and involve rapid timescales. We demonstrate the nascent approach of site-specific two-dimensional infrared (IR) spectroscopy to investigate the archetypical cytochrome P450, P450cam, to better delineate the mechanism of the lower regioselectivity of hydroxylation of the substrate norcamphor in comparison to the native substrate camphor. Specific locations are targeted throughout the enzyme by selectively introducing cyano groups that have frequencies in a spectrally isolated region of the protein IR spectrum as local vibrational probes. Linear and two-dimensional IR spectroscopy were applied to measure the heterogeneity and dynamics at each probe and investigate how they differentiate camphor and norcamphor recognition. The IR data indicate that the norcamphor complex does not fully induce a large-scale conformational change to a closed state of the enzyme adopted in the camphor complex. Additionally, a probe directed at the bound substrate experiences rapidly interconverting states in the norcamphor complex that explain the hydroxylation product distribution. Altogether, the study reveals large- and small-scale structural heterogeneity and dynamics that could contribute to selectivity of a cytochrome P450 and illustrates the approach of site-selective IR spectroscopy to elucidate protein dynamics.