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

• Published in: Chemphyschem Vol. 14; no. 18; pp. 4094 - 4101
• Main Authors: Pievo, Roberta, Angerstein, Brigitta, Fielding, Alistair J., Koch, Christian, Feussner, Ivo, Bennati, Marina
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 16.12.2013; WILEY‐VCH Verlag; Wiley; Wiley Subscription Services, Inc
• Subjects: Analytical chemistry; Chemistry; Electron Spin Resonance Spectroscopy; enzymatic reactions; Enzymes - chemistry; Enzymes - metabolism; EPR spectroscopy; Exact sciences and technology; Freezing; General and physical chemistry; Kinetics; Oxygenases - chemistry; Oxygenases - metabolism; radical intermediates; rapid freeze quench (RFQ); Spectrum analysis; Substrate Specificity; Time Factors; tyrosyl; enzymatic reactions; tyrosyl; Investigation method; EPR spectrometry; rapid freeze quench (RFQ); radical intermediates; EPR spectroscopy; Enzymatic reaction
• ISSN: 1439-4235; 1439-7641
• DOI: 10.1002/cphc.201300714

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. A powerful combination! The performance of a new micro rapid freeze‐quench apparatus combined with electron paramagnetic resonance (EPR) spectroscopy is reported, and the ease of setting up a multi‐frequency EPR analysis to investigate the kinetics and mechanisms of enzymatic processes is demonstrated.