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Cryoradiolysis and Cryospectroscopy for Studies of Heme-Oxygen Intermediates in Cytochromes P450

  • I. G. Denisov
  • Y. V. Grinkova
  • S. G. SligarEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 875)

Abstract

Cryogenic radiolytic reduction is one of the most straightforward and convenient methods of generation and stabilization of reactive iron–oxygen intermediates for mechanistic studies in chemistry and biochemistry. The method is based on one-electron reduction of the precursor complex in frozen solution via exposure to the ionizing radiation at cryogenic temperatures. Such approach allows for accumulation of the fleeting reactive complexes which otherwise could not be generated at sufficient amount for structural and mechanistic studies. Application of this method allowed for characterizing of peroxo-ferric and hydroperoxo-ferric intermediates, which are common for the oxygen activation mechanism in cytochromes P450, heme oxygenases, and nitric oxide synthases, as well as for the peroxide metabolism by peroxidases and catalases.

Key words

Radiolysis Radiolytic reduction Peroxo-ferric complex Hydroperoxo-ferric complex Cytochrome P450 Low-temperature methods 

Notes

Acknowledgments

We gratefully acknowledge the contribution into development of these methods and collaboration with Drs. T.M. Makris, I. Schlichting, B.M. Hoffman, R.M. Davydov, M. Ikeda-Saito, J.R. Kincaid, and P.J. Mak, much of which resulted in the cited works. We appreciate the help provided by Dr. S. Toshkov at the Nuclear Radiation Lab, University of Illinois, Urbana-Champaign, and Dr. J. Bentley while using the 60Co source in the Notre Dame Radiation Laboratory (Notre Dame University, IN). Irradiations were conducted partly at the Notre Dame Radiation Laboratory, which is a facility of the U.S. Department of Energy, Office of Basic Energy Sciences. This work is supported by NIH grants GM31756 and GM33775 to S.G.S.

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© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • I. G. Denisov
    • 1
  • Y. V. Grinkova
    • 1
  • S. G. Sligar
    • 1
    Email author
  1. 1.Department of Biochemistry, School of Molecular and Cellular BiologyUniversity of IllinoisUrbanaUSA

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