Solution 1H, 15N NMR spectroscopic characterization of substrate-bound, cyanide-inhibited human heme oxygenase:: Water occupation of the distal cavity
Solution 1H, 15N NMR spectroscopic characterization of substrate-bound, cyanide-inhibited human heme oxygenase:: Water occupation of the distal cavity
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DOI：
10.1021/ja036176t
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发表时间：
2003-11-05
影响因子：
15
通讯作者：
La Mar, GN
中科院分区：
文献类型：
--
作者：
Li, YM;Syvitski, RT;La Mar, GN
的氰化物抑制，基板结合的复杂的均匀N-15标记的人类血红素加氧酶，hHO的溶液NMR光谱研究，导致表征的活性位点的性质和身份的强氢键和有序的水分子内的氢键网络和芳香族簇的远端侧的占领。{H-1-N-15}-HSQC光谱证实了几个关键供体在特别稳健的H-键中的功能性，并且{H-1-N-15}HSQC-NOESY光谱导致鉴定了三个额外的稳健H-键，以及检测到两个相对较强的H-键，其身份无法确定。3D NMR实验仅提供了一个适度的，但重要的，扩展的分配，因为损失的关键TOCSY交叉峰由于线加宽的动态异质性在活性位点。稳态NOES饱和后的水信号定位在紧邻的氢键供体，其中六个很容易识别的晶体结构中的9个有序的水分子。另外三个定位在可用空间中，以解释观察到的NOES。饱和水共振时的N-15-过滤的稳态NOES和N-15-过滤的NOESY光谱表明水分子和五个芳环的质子之间存在显著的负NOES。许多NOES可以通过位于晶体结构中的水分子来合理化，但是强的水NOES，特别是对于Phe 47和Trp 96的环，需要在这些环附近存在至少另外两个固定的水分子。H-键网络的功能似乎是为了水分子提供稳定的氢过氧中间体，并作为一个管道的活性位点所需的9个质子每HO营业额。
A solution NMR spectroscopic study of the cyanide-inhibited, substrate-bound complex of uniformly N-15-labeled human heme oxygenase, hHO, has led to characterization of the active site with respect to the nature and identity of strong hydrogen bonds and the occupation of ordered water molecules within both the hydrogen bonding network and an aromatic cluster on the distal side. {H-1-N-15}-HSQC spectra confirm the functionalities of several key donors in particularly robust H-bonds, and {H-1-N-15}HSQC-NOESY spectra lead to the identification of three additional robust H-bonds, as well as the detection of two more relatively strong H-bonds whose identities could not be established. The 3D NMR experiments provided only a modest, but important, extension of assignments because of the loss of key TOCSY cross-peaks due to the line broadening from a dynamic heterogeneity in the active site. Steady-state NOES upon saturating the water signal locate nine ordered water molecules in the immediate vicinity of the H-bond donors, six of which are readily identified in the crystal structure. The additional three are positioned in available spaces to account for the observed NOES. N-15-filtered steady-state NOES upon saturating the water resonances and N-15-filtered NOESY spectra demonstrate significant negative NOES between water molecules and the protons of five aromatic rings. Many of the NOES can be rationalized by water molecules located in the crystal structure, but strong water NOES, particularly to the rings of Phe47 and Trp96, demand the presence of at least an additional two immobilized water molecules near these rings. The H-bond network appears to function to order water molecules to provide stabilization for the hydroperoxy intermediate and to serve as a conduit to the active site for the nine protons required per HO turnover.