Thesis (M.S.)--University of Missouri-Columbia, 2004. / Typescript. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1966. / Typescript. Vita. Description based on print version record. Includes bibliographical references (156-162).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1965. / Typescript. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1963. / Typescript. Abstracted in Dissertation abstracts, v. 23 (1963) no. 9, p. 3114. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 86-101).

Thesis (M.S.)--University of Wisconsin--Madison, 1967. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The prosthetic groups of the respiratory fumarate reductase from Escherichia coli have been studied by electron paramagnetic resonance. The iron-sulphur clusters of this enzyme were characterised in membranes from a strain of E. coli with amplified expression of the fumarate reductase. Two ferredoxin centres, paramagnetic in the reduced state (FR1 & FR2, Em -50mV & -280mV) were shown to be present at the same concentration as the flavin, together with a centre paramagnetic in the oxidised state (FR3, Em -30mV), present at the same concentration. Another ferredoxin signal was observed in reduced membranes at 1/10th the concentration of the other centres. The relaxation processes of the iron-sulphur centres were characterised and shown to be similar to those reported for other iron-sulphur centres. These relaxation processes changed when more than one centre was paramagnetic, indicating interaction between the centres, which were characterised between FR1 & FR2, and FR1 & FR3, by the observed changes in e.p.r. properties. Estimates of the distances between centres were made from these observed changes. The orientation of the g-tensors of the iron-sulphur centres was studied in membrane multilayers, both from a wild-type strain and a strain with amplified expression of the enzyme. The iron-sulphur clusters were shown to have distinct orientations in both cases, with the amplified strain producing crystalline multilayers. The interactions between the iron-sulphur centres were shown to have an angular dependence and thus to be magnetic dipole-dipole interactions. The location of the iron-sulphur centres was studied using the exogenous paramagnetic probe dysprosium(III), and they were all shown to be on the cytoplasmic aspect of the cell membrane. The catalytic site of fumarate reduction was also located as cytoplasmic, by the use of mutant strains of coli, and inhibitors of the dicarboxylic acid porter. The iron-sulphur centres were shown to be located deep within the catalytic subunits of the enzyme. The flavin moiety of fumarate reductase was characterised in the isolated enzyme by e. p. r. The semiquinone form of the flavin was shown to be stable in the physiological pH range and to have an Em7 of -12mV (n = 2). Interaction between the semiquinone and FR1 was shown and characterised.

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.

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