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Title: Preparation and characterisation of the NADH:ubiquinone oxidoreductases (complexes I) from three fungal species
Author: Bridges, H. R.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2010
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There are few eukaryotic model organisms established for the study of complex I. The first part of this thesis describes studies of complex I from one of them, Yarrowia lipolytica.  The intact enzyme was analysed using EPR spectroscopy, mass spectrometry and Mössbauer spectroscopy (by incorporation of 57Fe). The Iλ subcomplex of Y. lipolytica complex I was prepared by adapting the established protocol from Bos taurus. It comprises the hydrophilic enzyme domain which contains all the redox cofactors, and it was characterised by EPR spectroscopy and mass spectrometry. Conditions under which the subcomplex remained stable for several days were identified; crystallization trials were undertaken, but were ultimately unsuccessful. A summary of the most promising conditions is presented, along with a description of those factors which affect the stability and integrity of the protein. Two candidate organisms for the development of new model systems to study complex I were identified: Pichia pastoris and Pichia angusta. Protocols were developed for the preparation and purification of highly pure and catalytically active complex I from both species. Complex I from P. pastoris was particularly active, and proton translocation was observed. Like complex I from B. taurus, the Pichia enzymes produce reactive oxygen species in the form of superoxide, but at a higher level. Finally, mass spectrometry was used to fully characterise the protein composition of complex I from P. pastoris. 39 Subunits were identified, and a comparison of the compositions of the P. pastoris, Y. lipolytica and mammalian enzymes is presented. Complex I from P. pastoris is a robust, new model system for future structural and functional studies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available