Enzyme activation and catalysis: characterisation of the vibrational modes of substrate and product in protochlorophyllide oxidoreductase.
Enzyme activation and catalysis: characterisation of the vibrational modes of substrate and product in protochlorophyllide oxidoreductase.
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酶活化和催化：原叶绿素内酯氧化还原酶中底物和产物振动模式的表征。
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发表时间：
2011
期刊：
影响因子：
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通讯作者：
M. Groot
中科院分区：
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作者：
O. Sytina;M. Alexandre;D. Heyes;C. Hunter;B. Robert;R. van Grondelle;M. Groot
原叶绿素的光依赖性还原是叶绿素合成的关键步骤，由原叶绿素氧化还原酶（POR）催化，需要两个光子（O。A. Sytina等人，Nature，2008，456，1001-1008）。第一个光子激活酶-底物复合物，随后的第二个光子通过触发催化中间体的形成来启动光化学反应。这两个事件的特点是在红外光谱区的不同光谱变化。在这里，我们研究了POR结合和未结合底物以及产物的振动频率，从而提供了与PChlide：NADPH：TyrOH催化转化为Chlide：NADP（+）：TyrO（-）相关的1800-1250 cm（-1）区域光谱变化的详细归属。与未结合的Pchlide相比，POR结合的Pchlide的荧光线窄化光谱显示C=O酮基向下移动超过20 cm（-1）至相对较低的振动频率1653 cm（-1），表明发色团与蛋白质的结合通过强氢键发生。的C=C振动模式的频率是一致的六配位状态的POR结合Pchlide，这表明有两个协调的发色团和蛋白质残基，和/或水分子的中心Mg原子之间的相互作用。Chlide的C=C振动模式的频率与五配位状态一致，表明发色团的中心Mg原子与水分子之间存在单一相互作用。在4 cm（-1）光谱分辨率下对Pchlide：POR：NADPH复合物进行快速扫描FTIR测量，发现在1670 cm（-1）区域有一个新的谱带。酶活化阶段的FTIR光谱表明参与的核苷酸结合结构基序，并增加暴露的蛋白质活化后的溶剂。
The light-dependent reduction of protochlorophyllide, a key step in the synthesis of chlorophyll, is catalyzed by the enzyme protochlorophyllide oxidoreductase (POR) and requires two photons (O. A. Sytina et al., Nature, 2008, 456, 1001-1008). The first photon activates the enzyme-substrate complex, a subsequent second photon initiates the photochemistry by triggering the formation of a catalytic intermediate. These two events are characterized by different spectral changes in the infra-red spectral region. Here, we investigate the vibrational frequencies of the POR-bound and unbound substrate, and product, and thus provide a detailed assignment of the spectral changes in the 1800-1250 cm(-1) region associated with the catalytic conversion of PChlide:NADPH:TyrOH into Chlide:NADP(+):TyrO(-). Fluorescence line narrowed spectra of the POR-bound Pchlide reveal a C=O keto group downshifted by more than 20 cm(-1) to a relatively low vibrational frequency of 1653 cm(-1), as compared to the unbound Pchlide, indicating that binding of the chromophore to the protein occurs via strong hydrogen bond(s). The frequencies of the C=C vibrational modes are consistent with a six-coordinated state of the POR-bound Pchlide, suggesting that there are two coordination interactions between the central Mg atom of the chromophore and protein residues, and/or a water molecule. The frequencies of the C=C vibrational modes of Chlide are consistent with a five-coordinated state, indicating a single interaction between the central Mg atom of the chromophore and a water molecule. Rapid-scan FTIR measurements on the Pchlide:POR:NADPH complex at 4 cm(-1) spectral resolution reveal a new band in the 1670 cm(-1) region. The FTIR spectra of the enzyme activation phase indicate involvement of a nucleotide-binding structural motif, and an increased exposure of the protein to solvent after activation.