The anaerobic fumarate reductase of 'Escherichia Coli' : a study of its prosthetic groups
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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.
Thesis, PhD Doctor of Philosophy
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