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Multifrequency Pulsed Electron Paramagnetic Resonance on Metalloproteins

机译:金属蛋白的多频脉冲电子顺磁共振

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Metalloproteins often contain metal centers that are paramagnetic in some functional state of the protein; hence elec-ntron paramagnetic resonance (EPR) spectroscopy can be a powerful tool for studying protein structure and function.nDipolar spectroscopy allows the determination of the dipole dipole interactions between metal centers in protein com-nplexes, revealing the structural arrangement of different paramagnetic centers at distances of up to 8 nm. Hyperfine spec-ntroscopy can be used to measure the interaction between an unpaired electron spin and nuclear spins within a distance ofn0.8 nm; it therefore permits the characterization of the local structure of the paramagnetic center’s ligand sphere with verynhigh precision. In this Account, we review our laboratory’s recent applications of both dipolar and hyperfine pulsed EPRnmethods to metalloproteins.nWe used pulsed dipolar relaxation methods to investigate the complex of cytochrome c and cytochrome c oxidase, a non-ncovalent protein protein complex involved in mitochondrial electron-transfer reactions. Hyperfine sublevel correlation spec-ntroscopy (HYSCORE) was used to study the ligand sphere of iron sulfur clusters in complex I of the mitochondrial respiratorynchain and substrate binding to the molybdenum enzyme polysulfide reductase. These examples demonstrate the potentialnof the two techniques; however, they also highlight the difficulties of data interpretation when several paramagnetic spe-ncies with overlapping spectra are present in the protein. In such cases, further approaches and data are very useful to enhancenthe information content.nRelaxation filtered hyperfine spectroscopy (REFINE) can be used to separate the individual components of overlappingnparamagnetic species on the basis of differences in their longitudinal relaxation rates; it is applicable to any kind of pulsednhyperfine or dipolar spectroscopy. Here, we show that the spectra of the iron sulfur clusters in complex I can be sepa-nrated by this method, allowing us to obtain hyperfine (and dipolar) information from the individual species. Furthermore,nperforming pulsed EPR experiments at different magnetic fields is another important tool to disentangle the spectral com-nponents in such complex systems. Despite the fact that high magnetic fields do not usually lead to better spectral separa-ntion for metal centers, they provide additional information about the relative orientation of different paramagnetic centers.nOur high-field EPR studies on cytochrome c oxidase reveal essential information regarding the structural arrangement ofnthe binuclear CuA center with respect to both the manganese ion within the enzyme and the cytochrome in thenprotein protein complex with cytochrome c.
机译:金属蛋白通常包含在蛋白质某些功能状态下为顺磁性的金属中心。因此,电子顺磁共振(EPR)光谱可以成为研究蛋白质结构和功能的强大工具。距离可达8 nm。超精细光谱学可用于测量不成对的电子自旋与核自旋之间在n0.8 nm距离内的相互作用。因此,它可以非常高精度地表征顺磁中心配体球的局部结构。在此帐户中,我们回顾了实验室在金属蛋白上偶极脉冲和超细脉冲EPRn方法的最新应用。转移反应。超精细亚相关光谱法(HYSCORE)用于研究线粒体呼吸链复合体I中铁硫簇的配体和底物与钼酶多硫化物还原酶的结合。这些例子说明了这两种技术的潜力。然而,当蛋白质中存在几个具有重叠光谱的顺磁现象时,它们也突出了数据解释的困难。在这种情况下,进一步的方法和数据对于增强信息含量非常有用。它适用于任何种类的脉冲超光谱或偶极光谱学。在这里,我们表明,通过这种方法可以分离复合物I中铁硫簇的光谱,从而使我们能够从单个物种中获得超细(和偶极)信息。此外,在不同磁场下执行脉冲EPR实验是在这种复杂系统中解开光谱成分的另一个重要工具。尽管事实上高磁场通常不会导致金属中心更好的光谱分离,但它们提供了有关不同顺磁中心相对取向的更多信息。核内CuA中心相对于酶中的锰离子和蛋白质与细胞色素c的复合物中的细胞色素的排列。

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