Nienhaus Karin, Deng Pengchi, Kriegl Jan M, Nienhaus G Ulrich
Department of Biophysics, University of Ulm, 89069 Ulm, Germany.
Biochemistry. 2003 Aug 19;42(32):9647-58. doi: 10.1021/bi034788k.
Using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures, we have studied CO binding to the heme and CO migration among cavities in the interior of sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation. Photoproduct intermediates, characterized by CO in different locations, were selectively enhanced by laser illumination at specific temperatures. Measurements were performed on the wild-type protein and a series of mutants (L104W, I107W, I28F, and I28W) in which bulky amino acid side chains were introduced to block passageways between cavities or to fill these sites. Binding of xenon was also employed as an alternative means of filling cavities. In all samples, photolyzed CO ligands were observed to initially bind at primary docking site B in the vicinity of the heme iron, from where they migrate to the secondary docking sites, the Xe4 and/or Xe1 cavities. To examine the relevance of these internal docking sites for physiological ligand binding, we have performed room-temperature flash photolysis on the entire set of proteins in the CO- and O(2)-bound form. Together with the cryospectroscopic results, these data provide a clear picture of the role of the internal sites for ligand escape from and binding to myoglobin.
利用傅里叶变换红外（FTIR）光谱结合低温下的温度导数光谱（TDS），我们研究了光解离后，一氧化碳（CO）与抹香鲸一氧化碳肌红蛋白（MbCO）内部腔室中血红素的结合以及CO在这些腔室之间的迁移。通过在特定温度下的激光照射，以不同位置的CO为特征的光产物中间体被选择性增强。对野生型蛋白和一系列突变体（L104W、I107W、I28F和I28W）进行了测量，在这些突变体中引入了庞大的氨基酸侧链以阻断腔室之间的通道或填充这些位点。氙的结合也被用作填充腔室的另一种方法。在所有样品中，观察到光解的CO配体最初结合在血红素铁附近的主要对接位点B，然后从那里迁移到次要对接位点，即Xe4和/或Xe1腔室。为了研究这些内部对接位点与生理配体结合的相关性，我们对所有处于CO结合态和O(2)结合态的蛋白质进行了室温闪光光解。结合低温光谱结果，这些数据清楚地描绘了内部位点在配体从肌红蛋白逃逸和与肌红蛋白结合过程中的作用。
