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Structural and functional studies of respiratory chain proteins using infrared spectroscopy

Marshall, DCA; (2007) Structural and functional studies of respiratory chain proteins using infrared spectroscopy. Doctoral thesis , UCL (University College London). Green open access

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Abstract

Attenuated total reflection Fourier transform infrared (FTIR) spectroscopy in the 4000-800 cm"1 range was applied to NADH:ubiquinone oxidoreductase (complex I) and cytochrome bc complex of the respiratory electron transfer chain. High signal:noise redox difference spectra were acquired using novel electrochemical and perfusion techniques to reversibly adjust protein redox state which, where appropriate, was monitored by simultaneous UV/visible spectroscopy. Interpretation of spectra was assisted by hydrogen-deuterium exchange, 15N labelling and redox titration. Model compound data, including protonation state/redox difference spectra of histidine, flavin mononucleotide and ferredoxins, were recorded and are presented as a comprehensive 'toolkit' for assisting in acquisition and interpretation of protein IR data in general. Electrochemically-induced difference spectra of bovine and Rhodobacter capsulatus cytochrome bc complex are comparable to published perfusion data but are at significantly higher signahnoise demonstrating the functionality of the electrochemical technique. Comparison of wild-type and E295V Rba. capsulatus difference spectra resulting from haem reduction/oxidation definitively rules out Glu-295 as the principal carboxylic acid residue responsive to haem b redox change. Full redox difference spectra of Yarrowia lipolytica and bovine complex I are similar to published E. coli transmission FTIR spectra where signals in the amide I and II region were interpreted as indictors of large scale conformational change. Comparison of complex I and ferredoxin difference spectra rules out this interpretation. Signals in the presented spectra are tentatively assigned to reduced flavin and to protonation state change of one or more histidine residues. In iron-sulphur centre N2 redox difference spectra, measured in intact complex I for the first time, signals from substrate ubiquinone/ubiquinol formation are clear a putative histidine protonation change signal is also present. Perfusion induced-difference spectra, using NADH as reductant, indicate that NADH does not reduce the full complement of iron-sulphur centres and no signals indicative of large scale conformational change were present.

Type: Thesis (Doctoral)
Title: Structural and functional studies of respiratory chain proteins using infrared spectroscopy
Identifier: PQ ETD:593421
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest. Third party copyright material has been removed from the ethesis. Images identifying individuals have been redacted or partially redacted to protect their identity.
URI: https://discovery.ucl.ac.uk/id/eprint/1446092
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