In the last 20 years, the use of electron paramagnetic resonance (EPR) has made a pronounced and lasting impact in the field of structural biology. The advantage of EPR spectroscopy over other structural techniques is its ability to target even minor conformational changes in any biomolecule or macromolecular complex, independent of its size or complexity, or whether it is in solution or in the cell during a biological or chemical reaction. Here, we focus on the use of EPR spectroscopy to study transmembrane transport and transcription mechanisms. We discuss experimental and analytical concerns when referring to studies of two biological reaction mechanisms, namely, transfer of copper ions by the human copper transporter hCtr1 and the mechanism of action of the Escherichia coli copper-dependent transcription factor CueR. Last, we elaborate on future avenues in the field of EPR structural biology.