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Title: Characterization of the catalytic mechanisms of Escherichia coli fumarate reductase and ubiquinal oxidase cytochrome-bd
Author: Moodie, Alan Donaldson
Awarding Body: University of St Andrews
Current Institution: University of St Andrews
Date of Award: 1991
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Escherichia coli respiratory enzymes fumarate reductase and ubiquinol-oxygen oxido-reductase cytochrome-bd have been investigated with the aim of determining their catalytic mechanisms. A rapid-freeze quench apparatus was developed for discontinuous pre-steady state spectral analysis of fumarate reductase prosthetic group electron transfer. The ram system includes the application of stainless steel HPLC parts for mixing. The novel feature of the apparatus is the cryostat which is safer and more convenient to use than its predecessors. The cryostat was designed for application to electron paramagnetic resonance studies but could easily be modified for use with other spectroscopic techniques. Test reactions determined the resolution of the apparatus in the low millisecond range. Reduction/oxidation events for the iron-sulphur centres FR1 and FR3 of fumarate reductase were not kinetically resolved in the low millisecond range (internal equilibration rapid). From data presented, FR1 and FR3 are thought to be kinetically competent for the predicted maximum turnover of the fumarate reductase benzyl viologen assay (30s-1, Simpkin, 1985). E.p.r. signals for FR2, the low potential iron-sulphur centre, were not observed in the pre-steady state or steady state and the role of this centre in electron transfer remains ambiguous. The data is consistent with a sequential model of electron transfer from menaquinol→FR3→FR1→flavin→fumarate. A dual, high potential/low potential, pathway for fumarate reductase (Cammack et al., 1986a,b) is not consistent with available data. Ligand binding studies for cytochrome-bd demonstrate carbon monoxide and nitric oxide ligation to both haem-d and haem-b595. Photodissociation affects for the carbon monoxide inhibited oxidase (oxygen electrode) and time dependent haem-NO formation (optical and e.p.r.), suggest a bimetallic site with two haems in close proximity. The catalytic mechanism for dioxygen reduction is proposed as a single dioxygen ligation between haem-b595 and haem-d with subsequent reduction by haem-b558 and quinol. A stabilized (bound) semiquinone in close proximity to haem-b558 is also detected.
Supervisor: Ingledew, W. John Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QP603.D4M7 ; Dehydrogenases