Deciphering Cu(II) EPR in biological buffers: Implications for copper protein studies.

Copper proteins are essential for many biological processes, mediating electron transfer and redox catalysis in biochemistry and playing key roles in human health. Electron paramagnetic resonance (EPR) spectroscopy is a powerful tool to probe Cu(II) centers in these proteins, but its application can be complicated by spurious signals arising from adventitious Cu(II) binding to buffers or affinity tags. Here, we systematically examined Cu(II) EPR signals from common biological buffers (Tris, HEPES, MOPS) over pH 6.0-8.0, as well as from a His affinity tag. We show that Tris coordinates Cu(II) across this pH range, whereas HEPES binds Cu(II) weakly at near neutral pH and MOPS essentially does not chelate Cu(II) and rather precipitates Cu(II). These buffer-dependent Cu(II) complexes produce distinct EPR signatures that can interfere with spectral interpretation and spin quantifications. Using two case studies-a bacterial multicopper oxidase (MnxEFG) and Drosophila lysyl oxidase (DmLOX), we show how buffer choices and purification tags influence the observed Cu EPR spectra. In MnxEFG, switching from HEPES to Tris eliminated adventitiously bound Cu(II) and revealed the true copper content and sites. In DmLOX, we identified an EPR signature of Cu(II) bound to the protein's His-tag, which could be minimized by appropriate refolding conditions. These findings highlight the importance of controlling solution chemistry during sample preparation and suggest practical strategies to obtain cleaner, more interpretable spectra. Overall, this work provides guidance for minimizing nonspecific Cu(II) binding and improving the reliability of EPR analysis in studies of copper proteins in biological systems.