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  A rapid freeze-quench setup for multi-frequency EPR spectroscopy of enzymatic reactions.

Pievo, R., Angerstein, B., Fielding, A. J., Koch, C., Feussner, I., & Bennati, M. (2013). A rapid freeze-quench setup for multi-frequency EPR spectroscopy of enzymatic reactions. ChemPhysChem, 14(18), 4094-4101. doi:10.1002/cphc.201300714.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0014-C6C3-C Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0027-CBA1-4
Genre: Journal Article

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Pievo, R.1, Author              
Angerstein, B.2, Author              
Fielding, A. J.1, Author              
Koch, C., Author
Feussner, I., Author
Bennati, M.1, Author              
Affiliations:
1Research Group of Electron Paramagnetic Resonance, MPI for biophysical chemistry, Max Planck Society, ou_578606              
2Research Group of Solid-State NMR, MPI for biophysical chemistry, Max Planck Society, ou_persistent35              

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 Abstract: Electron paramagnetic resonance (EPR) spectroscopy in combination with the rapid freeze-quench (RFQ) technique is a well-established method to trap and characterize intermediates in chemical or enzymatic reactions at the millisecond or even shorter time scales. The method is particularly powerful for mechanistic studies of enzymatic reactions when combined with high-frequency EPR (ν≥90 GHz), which permits the identification of substrate or protein radical intermediates by their electronic g values. In this work, we describe a new custom-designed micro-mix rapid freeze-quench apparatus, for which reagent volumes for biological samples as small as 20 μL are required. The apparatus was implemented with homemade sample collectors appropriate for 9, 34, and 94 GHz EPR capillaries (4, 2, and 0.87 mm outer diameter, respectively) and the performance was evaluated. We demonstrate the application potential of the RFQ apparatus by following the enzymatic reaction of PpoA, a fungal dioxygenase producing hydro(pero)xylated fatty acids. The larger spectral resolution at 94 GHz allows the discernment of structural changes in the EPR spectra, which are not detectable in the same samples at the standard 9 GHz frequency.

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Language(s): eng - English
 Dates: 2013-11-082013-12-16
 Publication Status: Published in print
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 Rev. Method: Peer
 Identifiers: DOI: 10.1002/cphc.201300714
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Title: ChemPhysChem
Source Genre: Journal
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Pages: - Volume / Issue: 14 (18) Sequence Number: - Start / End Page: 4094 - 4101 Identifier: ISSN: 1439-4235