As synchrotron light sources and optics deliver greater photon flux on samples, X-ray-induced photo-chemistry is increasingly encountered in X-ray absorption spectroscopy (XAS) experiments. The resulting problems are particularly pronounced for biological XAS experiments. This is because biological samples are very often quite dilute and therefore require signal averaging to achieve adequate signal-to-noise ratios, with correspondingly greater exposures to the X-ray beam. This paper reviews the origins of photo-reduction and photooxidation, the impact that they can have on active site structure, and the methods that can be used to provide relief from X-ray-induced photo-chemical artifacts.

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SUMMARY Copper plays a critical role in prion protein (PrP) physiology. Cu2+ binds with high affinity to the PrP N-terminal octarepeat domain (OR), and intracellular copper promotes PrP expression. The molecular details of copper coordination within the OR are now well characterized. Here we examine how Cu2+ influences the interaction between the PrP N-terminal domain and the C-terminal globular domain. Using NMR and copper-nitroxide double electron-electron resonance (DEER) EPR, with molecular dynamics refinement, we localize the position of Cu2+ in its high-affinity OR-bound state. Our results reveal an interdomain cis interaction that is stabilized by a conserved, negatively charged pocket of the globular domain. Interestingly, this interaction surface overlaps an epitope recognized by the POM1 antibody, the binding of which drives rapid cerebellar degeneration mediated by the PrP N-terminus. The resulting structure suggests that the globular domain regulates the N-terminal domain by binding the Cu2+-occupied OR within a complementary pocket.

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Copper is a vital metal cofactor in enzymes that are essential to myriad biological processes. Cellular acquisition of copper is primarily accomplished through the Ctr family of plasma membrane copper transport proteins. Model peptide studies indicate that the human Ctr1 N-terminus binds to Cu(II) with high affinity through an amino terminal Cu(II), Ni(II) (ATCUN) binding site. Unlike typical ATCUN-type peptides, the Ctr1 peptide facilitates the ascorbate-dependent reduction of Cu(II) bound in its ATCUN site by virtue of an adjacent HH (bis-His) sequence in the peptide. It is likely that the Cu(I) coordination environment influences the redox behavior of Cu bound to this peptide; however, the identity and coordination geometry of the Cu(I) site has not been elucidated from previous work. Here, we show data from NMR, XAS, and structural modeling that sheds light on the identity of the Cu(I) binding site of a Ctr1 model peptide. The Cu(I) site includes the same bis-His site identified in previous work to facilitate ascorbate-dependent Cu(II) reduction. The data presented here are consistent with a rational mechanism by which Ctr1 provides coordination environments that facilitate Cu(II) reduction prior to Cu(I) transport.