X-RAY INDUCED PHOTOREDUCTION OF REDOX ACTIVE METAL IONS
The use of synchrotron light sources has provided insights into structure, function, and mechanism of numerous proteins and enzymes. The fields of macromolecular crystallography and (MX) X-ray absorption spectroscopy (XAS) in particular have developed and grown with the use of synchrotrons, and cryoprotection has been an invaluable tool in making advances in these fields. Cryoprotectants have been employed to mitigate damage to biological samples caused by ice crystal formation during the flash cooling process. In addition, cryoprotectants are useful by allowing cryogenic temperatures to be utilized thereby reducing thermal vibrations in XAS experiments, and thermal stress on crystals used in MX experiments. X-ray induced photochemistry is an increasingly encountered problem for XAS experiments given the use of powerful synchrotron light sources with increased photon flux. As photon flux has increased, there has been a corresponding increase in X-ray induced photoxidation and photoreduction artifacts observed in XAS experiments. Increases in flux density have also been shown to have a negative impact on active site structure determination in MX experiments. Initial XAS studies of the prion protein (PrP) octarepeat region (OR) with Cu(II) revealed changes in the near edge region of the X-ray absorption spectra. After as little as one sweep of OR peptide bound to Cu(II), changes in the near edge region of the spectrum showed the loss of Cu(II) and an increase of Cu(I). Analysis revealed the complete loss of Cu(II) in situ and formation of Cu(I) bound to the PrP OR upon longer exposure to X-rays. To examine this phenomenon, samples of 2 mM CuCl2 with various cryoprotectants commonly used in XAS and protein crystal experiments were used in order to delineate the cause of the observed photoreduction. The preliminary result indicated that glycerol was the causative agent in exacerbating the X-ray induced photo-reduction. A series of experiments was then designed to examine the effects of glycerol concentration, copper concentration, and flux density on photoreduction rates. Results of these experiments show a linear relationship between the rate of photo-reduction and the X-ray flux density. Having observed photoreduction in aqueous copper samples, experiments were designed and carried out to exploit this phenomenon to produce chemically interesting mercury species. Given the previously observed relative stability of mercury at lower flux density values, the creation of Hg(0) species proved intriguing and relatively simple compared to other methodologies. Reports of peripheral sources of electrons contributing to photoreduction in experiments proved an intriguing addition to the photoreduction studies reported herein. Having studied photoreduction exacerbated by common cryoprotectants, studies were carried out with additional sources of electrons to observe any contributions made to photoreduction, including those present in common buffers used in both crystallographic and X-ray absorption spectroscopy experiments. Following examination of the increase in the rate of photoreduction with the presence of common cryoprotectants, experiments were designed and carried out to ameliorate the effects of photoreduction. Ketone groups have been long known to be excellent scavengers of free radicals and were examined to observe their presumptive protective effect on X-ray induced photoreduction.
X-ray Absorption Spectroscopy, Photoreduction, Copper
Doctor of Philosophy (Ph.D.)