title: A rapid freeze-quench setup for multi-frequency EPR spectroscopy of enzymatic reactions.
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.
authors: Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen (Germany). rpievo@gwdg.de.
authors: Roberta
authors: R
authors: Pievo
authors: Brigitta
authors: B
authors: Angerstein
authors: Alistair J
authors: AJ
authors: Fielding
authors: Christian
authors: C
authors: Koch
authors: Ivo
authors: I
authors: Feussner
authors: Marina
authors: M
authors: Bennati
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism*; D005615:Freezing; D007700:Kinetics; D010105:Oxygenases / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D004798:Enzymes; D010105:Oxygenases
keywords: EPR spectroscopy; enzymatic reactions; radical intermediates; rapid freeze quench (RFQ); tyrosyl