Effective coupling of rapid freeze-quench to high-frequency electron paramagnetic resonance

Abstract

We report an easy, efficient and reproducible way to prepare Rapid-Freeze-Quench samples in sub-millimeter capillaries and load these into the probe head of a 275 GHz Electron Paramagnetic Resonance spectrometer. Kinetic data obtained for the binding reaction of azide to myoglobin demonstrate the feasibility of the method for high-frequency EPR. Experiments on the same samples at 9.5 GHz show that only a single series of Rapid-Freeze-Quench samples is required for studies at multiple microwave frequencies.

Performance Comparison of different Rapid Freeze-Quench Strategies for Electron Paramagnetic Resonance

“…The most common procedure to test the performance of the RFQ coupled with EPR is using the azidebinding reaction to ferric myoglobin (Mb) [2,6,15,22]. In ferric Mb or aquomet Mb the heme iron is bound to a histidine residue on the proximal side and a water molecule on the distal side, leading to an EPR signal characteristic of a high-spin (HS) Fe(III) heme form (Figure S3 in the supplementary material).…”

“…A similar approach has been used by Kaufmann et al [13] to prepare freezequenched samples for W-band EPR, but with their setup it was possible to collect samples quenched at different times in a single freezing experiment. Finally, the group of E. Groenen showed an improved way of collecting freeze-quench particles from isopentane [14] and demonstrated that it allows an effective coupling of RFQ with high-frequency EPR [15]. Moreover, they demonstrated that only a single series of RFQ samples is needed for multi-frequency EPR (9.5 GHz and 275 GHz).…”

Spatiotemporal Resolution of Conformational Changes in Biomolecules by Combining Pulsed Electron–Electron Double Resonance Spectroscopy with Microsecond Freeze-Hyperquenching

“…Calibration of the MHQ Device. The reaction kinetics between equine heart metmyoglobin (MetMb) and sodium azide (NaN 3 ) 55 was employed to calibrate the MHQ aging times. In the apo state, the Fe(III) ion in the heme group is in the high-spin state (S = 5/2, abbreviated hs), and binding of azide switches it to the low-spin state (S = 1/2, abbreviated ls).…”