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.
journal_name
J Inorg Biochemjournal_title
Journal of inorganic biochemistryauthors
Wright JJ,Salvadori E,Bridges HR,Hirst J,Roessler MMdoi
10.1016/j.jinorgbio.2016.04.025subject
Has Abstractpub_date
2016-09-01 00:00:00pages
201-206eissn
0162-0134issn
1873-3344pii
S0162-0134(16)30111-8journal_volume
162pub_type
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journal_title:Journal of inorganic biochemistry
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