Development of Rapid Freeze-Quench Sample Preparation Techniques for Use in High Frequency Electron Paramagnetic Resonance

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This award will support work whose objective is to permit the use of rapid freeze-quench (RFQ) sample preparation with high frequency electron paramagnetic resonance and electron-nuclear double resonance (HF-EPR/ENDOR) spectroscopy. While the RFQ technique is extensively used in conjunction with conventional low frequency EPR/ENDOR spectrometers, technical difficulties have prevented its optimal use at high frequencies. RFQ arrests the reaction of enzymes on the millisecond time scale to facilitate the investigation of short-lived catalytic intermediate species. The technique involves rapidly mixing two reactants (e. g. an enzyme and its substrate) and freezing the resulting mixture within a known time period (from 5 - 1000 milliseconds) by spraying it into a cryogenic bath. However, the small wavelengths and correspondingly small sample tubes and resonant cavities commonly used for high-frequency EPR make the application of RFQ techniques difficult, and have thus prevented the study of RFQ samples with this spectroscopic technique. The objective will be pursued via two general routes. One is the modification of existing RFQ by adapting them to the small HF-EPR capillary sample tubes. The other, larger effort will be devoted to modification of high frequency EPR instrumentation so that it is compatible with existing RFQ technology. This will involve the development of overmoded resonant EPR cavities that accept the standard, large RFQ sample tubes. These tubes are currently prepared in most laboratories that use low frequency EPR. Both approaches - the adaptation of RFQ techniques to high-frequency capillary tubes and the development of overmoded resonant cavities that accept standard RFQ tubes - will permit the study of short-lived intermediates in normal enzymatic reactions. Prior to the development of RFQ techniques, standard strategies for such studies involved the use of inhibitors or substrate analogues that arrested reactions at an intermediate stage. While such intermediates can provide important structural information about enzymes, they are less useful in providing information about electronic properties that are key to understanding the enzyme's role in the reaction.