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

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. Green open access

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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.

Type: Article
Title: Small-volume potentiometric titrations: EPR investigations of Fe-S cluster N2 in mitochondrial complex
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.jinorgbio.2016.04.025
Publisher version: http://dx.doi.org/10.1016/j.jinorgbio.2016.04.025
Language: English
Additional information: © 2016 The Author. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Science & Technology, Life Sciences & Biomedicine, Physical Sciences, Biochemistry & Molecular Biology, Chemistry, Inorganic & Nuclear, Chemistry, Electron Paramagnetic Resonance, Iron-Sulfur Clusters, Nadh:ubiquinone Oxidoreductase, Redox Titrations, Iron-Sulfur Clusters, Buffer Solutions, Spectroscopy, Nadh, Oxidoreductase, Resonance, Ph, Spectra, Enzyme
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > London Centre for Nanotechnology
URI: http://discovery.ucl.ac.uk/id/eprint/1542737
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