New pulsed EPR methods and their application to characterize mitochondrial complex I |
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Authors: | Thorsten Maly Adrian Cernescu Thomas F. Prisner |
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Affiliation: | a Francis Bitter Magnet Laboratory and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA b Molecular Bioenergetics Group, Cluster of Excellence Frankfurt “Macromolecular Complexes”, Medical School, Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany c Institut für Physikalische und Theoretische Chemie, Johann Wolfgang Goethe-Universität Frankfurt, D-60439 Frankfurt am Main, Germany |
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Abstract: | Electron Paramagnetic Resonance (EPR) spectroscopy is the method of choice to study paramagnetic cofactors that often play an important role as active centers in electron transfer processes in biological systems. However, in many cases more than one paramagnetic species is contributing to the observed EPR spectrum, making the analysis of individual contributions difficult and in some cases impossible. With time-domain techniques it is possible to exploit differences in the relaxation behavior of different paramagnetic species to distinguish between them and separate their individual spectral contribution. Here we give an overview of the use of pulsed EPR spectroscopy to study the iron-sulfur clusters of NADH:ubiquinone oxidoreductase (complex I). While FeS cluster N1 can be studied individually at a temperature of 30 K, this is not possible for FeS cluster N2 due to its severe spectral overlap with cluster N1. In this case Relaxation Filtered Hyperfine (REFINE) spectroscopy can be used to separate the overlapping spectra based on differences in their relaxation behavior. |
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Keywords: | BDPA, α,γ-Bisdiphenylene-β-phenylallyl BDPA/PS, BDPA dissolved in polystyrene Complex I, NADH:ubiquinone oxidoreductase CuHis, Copper Histidine Complex DOSY, Diffusion Ordered Spectroscopy ENDOR, Electron Nuclear Double Resonance EPR, Electron Paramagnetic Resonance ESEEM, Electron Spin Echo Envelope Modulation FeS, Iron-sulfur HYSCORE, Hyperfine Sublevel Correlation iLT, inverse Laplace transformation NADH, Nicotineamide Adenine Dinucleotide NMR, Nuclear Magnetic Resonance PELDOR, Pulsed Electron Double Resonance REFINE, Relaxation Filtered Hyperfine ROS, Reactive Oxygen Species TEMPO, 2,2,6,6-Tetramethylpiperidine-1-oxyl TEMPO/PS, TEMPO dissolved in polystyrene |
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