Small-volume potentiometric titrations: EPR investigations of Fe-S cluster N2 in mitochondrial complex I
File(s)
Published version
Author(s)
Wright, John J
Salvadori, Enrico
Bridges, Hannah R
Hirst, Judy
Roessler, Maxie M
Type
Journal Article
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.
Date Issued
2016-09-01
Date Acceptance
2016-04-18
Citation
Journal of Inorganic Biochemistry, 2016, 162, pp.201-206
ISSN
0162-0134
Publisher
Elsevier
Start Page
201
End Page
206
Journal / Book Title
Journal of Inorganic Biochemistry
Volume
162
Copyright Statement
© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
Identifier
http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000388059600023&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=1ba7043ffcc86c417c072aa74d649202
Subjects
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
Publication Status
Published
Date Publish Online
2016-04-20