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A Rapid Freeze-Quench Setup for Multi-Frequency EPR Spectroscopy of Enzymatic Reactions

Authors

  • Dr. Roberta Pievo,

    Corresponding author
    1. Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen (Germany)
    • Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen (Germany)

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  • Brigitta Angerstein,

    1. Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen (Germany)
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  • Dr. Alistair J. Fielding,

    1. Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen (Germany)
    2. Photon Science Institute, the University of Manchester, Oxford Road, M13 9PL Manchester (United Kingdom)
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  • Dr. Christian Koch,

    1. Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University, Justus-von-Liebig-Weg 11, 37077 Göttingen (Germany)
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  • Prof. Dr. Ivo Feussner,

    1. Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University, Justus-von-Liebig-Weg 11, 37077 Göttingen (Germany)
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  • Prof. Dr. Marina Bennati

    Corresponding author
    1. Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen (Germany)
    2. Department of Chemistry, Georg-August-University, Tammannstraße 2, 37077 Göttingen (Germany)
    • Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen (Germany)

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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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