Electron paramagnetic resonance study of radiation damage in photosynthetic reaction center crystals.
Electron paramagnetic resonance (EPR) was used to simultaneously study radiation-induced cofactor reduction and damaging radical formation in single crystals of the bacterial reaction center (RC). Crystals of Fe-removed/Zn-replaced RC protein from Rhodobacter ( R.) sphaeroides R26 were irradiated with varied radiation doses at cryogenic temperature and analyzed for radiation-induced free radical formation and alteration of light-induced photosynthetic electron transfer activity using high-field (HF) D-band (130 GHz) and X-band (9.5 GHz) EPR spectroscopies. These analyses show that the formation of radiation-induced free radicals saturated at doses 1 order of magnitude smaller than the amount of radiation at which protein crystals lose their diffraction quality, while light-induced RC activity was found to be lost at radiation doses at least 1 order of magnitude lower than the dose at which radiation-induced radicals exhibited saturation. HF D-band EPR spectra provide direct evidence for radiation-induced reduction of the quinones and possibly other cofactors. These results demonstrate that substantial radiation damage is likely to have occurred during X-ray diffraction data collection used for photosynthetic RC structure determination. Thus, both radiation-induced loss of photochemical activity in RC crystals and reduction of the quinones are important factors that must be considered when correlating spectroscopic and crystallographic measurements of quinone site structures.
Utschig
Lisa M
LM
Chemical Sciences and Engineering Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois 60439, USA.
Chemerisov
Sergey D
SD
Tiede
David M
DM
Poluektov
Oleg G
OG
D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005609:Free Radicals; D007501:Iron / Q000737:chemistry; D008958:Models, Molecular; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000528:radiation effects*; D011487:Protein Conformation; D012242:Rhodobacter sphaeroides / Q000378:metabolism; D014961:X-Ray Diffraction; D015032:Zinc / Q000737:chemistry
D005609:Free Radicals; D045322:Photosynthetic Reaction Center Complex Proteins; D007501:Iron; D015032:Zinc