Abstract
The combination of rapid freeze quenching (RFQ) with resonance Raman (RR) spectroscopy represents a unique tool with which to investigate the nature of short-lived intermediates formed during the enzymatic reactions of metalloproteins. Commercially available equipment allows trapping of intermediates within a millisecond to second time scale for low-temperature RR analysis resulting in the direct detection of metal–ligand vibrations and porphyrin skeletal vibrations in hemoproteins. This chapter briefly discusses RFQ-RR studies carried out previously in our laboratory and presents, as a practical example, protocols for the preparation of RFQ samples of the reaction of metmyoglobin with nitric oxide (NO) under anaerobic conditions. Also described are important controls and practical procedures for the analysis of these samples by low-temperature RR spectroscopy.
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Acknowledgments
This work was supported by the National Institute of Health (P.M.L., GM074785). H.M. acknowledges the financial support from the Japan Society for the Promotion of Science (Research Fellowship for Young Scientists).
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Matsumura, H., Moënne-Loccoz, P. (2014). Characterizing Millisecond Intermediates in Hemoproteins Using Rapid-Freeze-Quench Resonance Raman Spectroscopy. In: Fontecilla-Camps, J., Nicolet, Y. (eds) Metalloproteins. Methods in Molecular Biology, vol 1122. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-794-5_8
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DOI: https://doi.org/10.1007/978-1-62703-794-5_8
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