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

Area of Science:

• Biochemistry and Biophysics
• Bioinorganic Chemistry
• Spectroscopy

Background:

• Copper proteins are vital for biological electron transfer and redox catalysis, impacting human health.
• Electron paramagnetic resonance (EPR) spectroscopy is crucial for studying Cu(II) centers in proteins.
• Spurious Cu(II) signals from buffers and affinity tags can impede EPR spectral interpretation and quantification.

Purpose of the Study:

• To systematically investigate Cu(II) EPR signals from common biological buffers (Tris, HEPES, MOPS) and a His affinity tag.
• To assess the impact of buffer choice and purification tags on EPR spectra of copper proteins.
• To provide practical strategies for minimizing nonspecific Cu(II) binding in EPR studies.

Main Methods:

• Systematic examination of Cu(II) EPR signals across a pH range (6.0-8.0) using Tris, HEPES, and MOPS buffers.
• Analysis of Cu(II) binding to a His affinity tag.
• Case studies involving a bacterial multicopper oxidase (MnxEFG) and Drosophila lysyl oxidase (DmLOX) to evaluate buffer and tag effects on EPR spectra.

Main Results:

• Tris coordinates Cu(II) across the tested pH range; HEPES shows weak binding near neutral pH; MOPS precipitates Cu(II).
• Buffer-dependent Cu(II) complexes generate distinct EPR signatures, interfering with spectral analysis.
• Switching from HEPES to Tris in MnxEFG studies eliminated adventitious Cu(II), revealing true copper content.
• An EPR signature of Cu(II) bound to the His-tag in DmLOX was identified and could be minimized by refolding.

Conclusions:

• Buffer selection and control of solution chemistry are critical for accurate EPR analysis of copper proteins.
• Nonspecific Cu(II) binding can be minimized by choosing appropriate buffers (e.g., MOPS) and optimizing purification protocols.
• This study offers practical guidance for obtaining cleaner, more reliable EPR spectra in bioinorganic and biochemical research.