Can soaked-in scavengers protect metalloprotein active sites from reduction during data collection?
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.
Macedo
Sofia
S
Department for Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030 Vienna, Austria.
Pechlaner
Maria
M
Schmid
Walther
W
Weik
Martin
M
Sato
Katsuko
K
Dennison
Christopher
C
Djinović-Carugo
Kristina
K
D016477:Artifacts; D001400:Azurin / Q000737:chemistry / Q000528:radiation effects / Q000648:ultrastructure; D001665:Binding Sites; D018360:Crystallography, X-Ray / Q000379:methods*; D016166:Free Radical Scavengers / Q000737:chemistry*; D008667:Metalloproteins / Q000737:chemistry* / Q000528:radiation effects* / Q000648:ultrastructure; D009211:Myoglobin / Q000737:chemistry / Q000528:radiation effects / Q000648:ultrastructure; D010084:Oxidation-Reduction / Q000528:radiation effects; D011485:Protein Binding; D011487:Protein Conformation / Q000528:radiation effects; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012996:Solutions
D016166:Free Radical Scavengers; D008667:Metalloproteins; D009211:Myoglobin; D012996:Solutions; D001400:Azurin