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Title: Hydrogen as fuel for enteric bacteria : biochemistry of the membrane bound NiFe hydrogenase
Author: Flanagan, Lindsey
ISNI:       0000 0004 6062 670X
Awarding Body: University of York
Current Institution: University of York
Date of Award: 2016
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Hydrogen is considered both a fuel for the future and an ancient fuel for life. Hydrogenases catalyse the interconversion of molecular hydrogen, H2, and protons, H+. A subgroup of hydrogenases, the membrane bound NiFe hydrogenases (MBH), have been the subject of much research interest. This is firstly due to their possible applications in biotechnology, but also because they have been implicated in the virulence of gut pathogens. The MBH are divided into O2 tolerant and O2 sensitive based on their ability to catalyse H2 oxidation in the presence of O2, and the two classes are both structurally and mechanistically distinct. Understanding these distinctions is important both for technology which aims to achieve more minimal models of enzymes, but also for relating the way different MBH are expressed at different stages of infection. The properties of variants of two O2 tolerant MBH, Escherichia coli Hyd 1 and Salmonella enterica Hyd 5 and one O2 sensitive MBH, E. coli Hyd 2, have been examined with regards to how the properties of specific amino acids achieve control over catalysis and O2 tolerance. Red®/ET® recombination methodology has been applied for the first time to hydrogenases. This methodology allows the rapid generation of hydrogenase knockouts and single site variants in E. coli in addition to the incorporation of polyhistidine tags to enable protein purification. Purified native and variant hydrogenases have been studied with protein film electrochemistry. Hyd 1 and Hyd-5 E73A and H229A variants were shown to have diminished O2 tolerance whilst a Hyd 1 E73Q variant had an increased catalytic bias towards H2 production. It was established that Hyd 1 was expressed during growth in glucose limited minimal media, although no change in growth rate or competitive ability was seen in a Hyd 1 knockout strain.
Supervisor: Moir, James ; Parkin, Alison Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available