title: Small-volume potentiometric titrations: EPR investigations of Fe-S cluster N2 in mitochondrial complex I.
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=-158mV, 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.
authors.affiliation: School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, United Kingdom.
authors.forename: John J
authors.initials: JJ
authors.lastname: Wright
authors.affiliation: School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, United Kingdom; London Centre of Nanotechnology, University College London, 17-19 Gordon Street, London WC1H 0AH, United Kingdom.
authors.forename: Enrico
authors.initials: E
authors.lastname: Salvadori
authors.affiliation: Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, United Kingdom.
authors.forename: Hannah R
authors.initials: HR
authors.lastname: Bridges
authors.affiliation: Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, United Kingdom.
authors.forename: Judy
authors.initials: J
authors.lastname: Hirst
authors.affiliation: School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, United Kingdom. Electronic address: m.roessler@qmul.ac.uk.
authors.forename: Maxie M
authors.initials: MM
authors.lastname: Roessler
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D042967:Electron Transport Complex I / Q000737:chemistry*; D004583:Electrons; D007501:Iron / Q000737:chemistry*; D008928:Mitochondria / Q000737:chemistry* / Q000201:enzymology; D010084:Oxidation-Reduction; D011199:Potentiometry / Q000379:methods*; D015203:Reproducibility of Results; D013455:Sulfur / Q000737:chemistry*
chemical_list: D013455:Sulfur; D007501:Iron; D042967:Electron Transport Complex I
keywords: Electron paramagnetic resonance; Iron-sulfur clusters; NADH:ubiquinone oxidoreductase; Redox titrations