Can soaked-in scavengers protect metalloprotein active sites from reduction during data collection? (2009)

Author(s): Macedo S, Pechlaner M, Schmid W, Weik M, Sato K, Dennison C, Djinovic-Carugo K

    Abstract: One of the first events taking place when a crystal of a metalloprotein is exposed to X-ray radiation is photoreduction of the metal centres. The oxidation state of a metal cannot always be determined from routine X-ray diffraction experiments alone, but it may have a crucial impact on the metal's environment and on the analysis of the structural data when considering the functional mechanism of a metalloenzyme. Here, UV-Vis microspectrophotometry is used to test the efficacy of selected scavengers in reducing the undesirable photoreduction of the iron and copper centres in myoglobin and azurin, respectively, and X-ray crystallography to assess their capacity of mitigating global and specific radiation damage effects. UV-Vis absorption spectra of native crystals, as well as those soaked in 18 different radioprotectants, show dramatic metal reduction occurring in the first 60 s of irradiation with an X-ray beam from a third-generation synchrotron source. Among the tested radioprotectants only potassium hexacyanoferrate(III) seems to be capable of partially mitigating the rate of metal photoreduction at the concentrations used, but not to a sufficient extent that would allow a complete data set to be recorded from a fully oxidized crystal. On the other hand, analysis of the X-ray crystallographic data confirms ascorbate as an efficient protecting agent against radiation damage, other than metal centre reduction, and suggests further testing of HEPES and 2,3-dichloro-1,4-naphtoquinone as potential scavengers.

    Notes: Research Support, Non-U.S. Gov't Denmark

      • Date: 25-02-2009
      • Journal: Journal of Synchrotron Radiation
      • Volume: 16
      • Issue: 2
      • Pages: 191-204
      • Publisher: Wiley-Blackwell Munksgaard
      • Publication type: Article
      • Bibliographic status: Published

      Keywords: Artifacts Azurin/chemistry/radiation effects/ultrastructure Binding Sites Crystallography, X-Ray/methods Free Radical Scavengers/chemistry Metalloproteins/chemistry/radiation effects/ultrastructure Myoglobin/chemistry/radiation effects/ultrastructure Oxidation-Reduction/radiation effects Protein Binding Protein Conformation/radiation effects Reproducibility of Results Sensitivity and Specificity Solutions


      Professor Christopher Dennison
      Professor of Biological Chemistry