Copper participates in multiple key biological processes. It plays the role of cofactor in multiple enzymes i within the cell, where it is transported as Cu(I) by specific metallochaperones. The incorrect homeostasis of copper is related to diseases such as Wilson’s and Menkes’, and may have pernicious effects in neurodegenerative diseases such as Alzheimer’s. This is why understanding biological processes that involve copper, such as transmetallation between different proteins or peptides, and the interaction of these complexes with reducing agents with the purpose of transporting Cu inside the cell, and the dangerous reduction of Cu(II) to then produce reactive oxygen species (ROS) is of great relevance.
In this work, we aim to elucidate the mechanism of reduction of copper (II) to copper (I) with glutathione while bound to the naturally occurring peptide GHK, as a possible first step for his intracellular transport via the CTR1 membrane, which is a specific transporter of Cu(I). For this, we have measured UV-Vis data during the course of the reaction at several pH values, using a stopped-flow mixer. Our preliminary analysis demonstrate that the reduction reaction of the Cu(GHK) complex with glutathione gets completed in a matter of a few hundred miliseconds at pH values below 5, but becomes slower as the pH increases, favoring the presence of a long-lived intermediate which we have isolated at pH 8 to investigate its structure.
Furthermore, High-resolution X-ray absorption spectroscopy and EPR experiments were performed to characterize the geometry and electronic structure of this isolated intermediate. For the interpretation of the data, we have used a holistic method where optimized structural models need to fit all the available spectroscopic data. The elucidation of this key intermediate will help us to establish a reaction mechanism, and will shed light on possible routes for the complex copper transportation process into the cell. Furthermore, the obtained results demonstrate the applicability of this holistic approach to address similar questions for other dynamic processes in biological systems.