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.
Type: | Article |
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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 UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences |
URI: | https://discovery.ucl.ac.uk/id/eprint/1542737 |
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