Wright, JJ; Salvadori, E; Bridges, HR; Hirst, J; Roessler, MM; (2016) Small-volume potentiometric titrations: EPR investigations of Fe-S cluster N2 in mitochondrial complex. Journal of Inorganic Biochemistry , 162 pp. 201-206. 10.1016/j.jinorgbio.2016.04.025.

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Abstract

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.

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