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Title: The preparation and characterization of component C of Methylococcus capsulatus (Bath)
Author: Lund, John
ISNI:       0000 0001 3613 9926
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1983
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The methane monooxygenase (MHO) present in soluble extracts of Methylococcus capsulatus (Bath) can be resolved into three components, called protein A, protein B, and protein C. Protein C, the subject of this thesis, is a polypeptide chain with one FAD and one iron-sulphur centre per molecule, and is the reductase component of the monooxygenase system, for which NADH is the reducing substrate. Protein C transfers reducing equivalents to protein A, the monooxygenase component, responsible for substrate hydroxylation. The aim of this project was to investigate how electrons were transferred from NADH to protein A, and work focused on the 2 redox centres of protein C. (1) Protein C has been prepared and purified by a modified method, which is quicker and more reproducible than published methods. (2) A critical comparison of present and previous characterisation work on protein C has been undertaken. (3) The three aid-point potentials of the protein C redox centres have been determined and shown to be consistent with their ability to transfer electrons to protein A from NADH: additionally, the optical contributions of the redox centres in various states of reduction have been resolved. (4) The order of electron Flow in the two redox centres has been shown to be, NADH > FAD > FeS > protein A, by selective removal and reconstitution of the centres. (5) With saturating NADH, it has been shown that NADH interaction and reduction of protein C, and electron transfer to protein A, are not rate-limiting to the overall HHO reaction, and electron transfer to protein A is independent of protein B, which is proposed to act after this step; a gross kinetic mechanism is proposed. (6) Possible technological uses of protein C are explored, based on the ability of protein C to be reduced by redox dyes, to regenerate NADH, and to reduce the redox electrode without radiators.
Supervisor: Not available Sponsor: British Petroleum Company
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QP Physiology ; QR Microbiology