ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
SL2. X-ray Radiation Damage on Metallo-enzymes:
Can Soaked-in Scavengers Protect Metalloprotein Active Sites from
Reduction During Data Collection?
S. Macedo a , M. Pechlaner a , W. Schmid b , M. Weik c , K. Sato d , Ch. Dennison d ,
K. Djinović-Carugo a
a
Dept. for Biomolecular Structural Chemistry, Max F Perutz Laboratories, University of Vienna, Campus
Vienna Biocenter 5, 1030 Vienna, Austria
b
Institute of Organic Chemistry, University of Vienna, Währinger Straße 38, 1090 Vienna, Austria
c
Laboratoire de Biophysique Moléculaire, Institut de Biologie Structurale J.-P. Ebel, CEA CNRS UJF, UMR
5075, 41 rue Jules Horowitz, 38027 Grenoble Cedex 01, France
d
Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne, NE2
4HH, UK
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. A combination of UV-vis
micro-spectrophotometry and X-ray crystallography was used to test the efficacy of 18 selected scavengers in
reducing the undesirable photo-reduction of the iron and copper centres in myoglobin and azurin, respectively.
Results of the UV-vis absorption spectra show dramatic metal reduction occurring in the first 60 seconds of
irradiation with an X-ray beam from a 3 rd generation synchrotron source, and only two scavengers were capable
of partially mitigating the rate of metal photo-reduction, but not to a sufficient extent that would allow a
complete data set to be recorded from a fully oxidised 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.
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