Cu(II)-glycerol-N-ethylmorpholine complex stability revealed by X-ray spectroscopy

We report on a detailed X-ray absorption spectroscopy study of the Cu(II) coordination mode in aqueous solution in the presence of glycerol cryoprotectant and N-ethylmorpholine buffer. The first additive is often used when working at very low temperatures with the purpose of avoiding unwanted ice diffraction, the second is added to allow adjusting the pH at the desired value. Additives of this kind are routinely used in experiments. It is thus mandatory to have a precise understanding of their structural interaction with metals and in particular with Cu, which is among the most important protein-binding ions, in order to identify spurious spectral signatures in the X-ray spectrum of metal-protein complexes possibly polluting the collected data. Our XAS data show that the spectrum of Cu in the presence of these additives is significantly different from that of Cu in pure water, thus suggesting a direct interaction of glycerol and N-ethylmorpholine with the metal ion. Classical and ab initio numerical simulations are employed to construct structural models of the metal environment able to reproduce the features of the measured X-ray spectrum. In particular, starting from system configurations extracted from classical molecular dynamics simulations, we have successively refined their geometrical structure by optimizing the local Cu binding site by DFT relaxation runs. The fit to the experimental data obtained from the initial geometrical parameters of the Cu binding site determined in this way is very good and shows that both glycerol and N-ethylmorpholine contribute to the Cu coordination.