Small-volume potentiometric titrations: EPR investigations of Fe-S cluster N2 in mitochondrial complex

EPR-based potentiometric titrations are a well-established method for determining the reduction potentials of cofactors in large and complex proteins with at least one EPR-active state. However, such titrations require large amounts of protein. Here, we report a new method that requires an order of magnitude less protein than previously described methods, and that provides EPR samples suitable for measurements at both X- and Q-band microwave frequencies. We demonstrate our method by determining the reduction potential of the terminal [4Fe-4S] cluster (N2) in the intramolecular electron-transfer relay in mammalian respiratory complex I. The value determined by our method, Em7 = − 158 mV, is precise, reproducible, and consistent with previously reported values. Our small-volume potentiometric titration method will facilitate detailed investigations of EPR-active centres in non-abundant and refractory proteins that can only be prepared in small quantities.