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Studies on the succinate oxidase system of E. coli

University of British Columbia

The succinate oxidase system of E. coli has been studied from three main viewpoints: (a) the preparation and properties of succinate dehydrogenase (SDH), (b) the function of nonheme iron, and (c) the sequence of the components of the respiratory chain. Three different preparations containing SDH activity were isolated from this organism. These were the particulate fraction, soluble respiratory complex, and soluble SDH. The partial purification and characterization of these enzymes or complexes was performed. The particulate fraction consisted of membrane fragments which contained the whole respiratory chain and which oxidized succinate and NADH. The soluble respiratory complex contained both SDH and cytochrome b(1). The molecular weight was 1.6 x 10(6). The soluble SDH did not contain cytochrome b(1) and had a molecular weight of 100,000. One of the characteristic properties of SDH of the particulate fraction and the soluble respiratory complex was activation. If the enzyme was prepared in phosphate buffer both succinate oxidase and SDH activities could be activated by heating at 38° in the presence of succinate. The enzyme was stabilized by succinate in the absence of heating. Activation of succinate oxidase seemed to be mainly due to the activation of SDH. A second activation phenomenon which was independent of heat treatment was also observed. When the enzyme was prepared in Tris buffer with succinate the activated enzyme was formed at 0°. Heating did not further increase its activity. Activation by heat was irreversible. The heat-activated enzyme deactivated to a form which could not be reactivated. The heat-independent activated enzyme was more stable. The two activation phenomena thus seemed to be different. In contrast, the soluble SDH did not show the activation phenomenon nor was it stabilized by substrate. A mechanism for the activation of SDH is proposed. The nature, properties, role and location of nonheme iron in the particulate fraction of E. coli was investigated. The level of nonheme ferrous or ferric iron in the particulate fraction was determined spectrophotometrically using o-phenanthroline or Tiron. Analysis of iron by both chemical and spectrophotometric methods showed that only 45% of the total iron reacted with o-phenanthroline ("o-phenanthroline-reacting iron"). Heme iron constituted 5% of the total iron. The rest of total iron was not exposed by treating the particulate fraction with detergents or urea. The nature of the remainder of the total iron (50%) is unknown. Half of the o-phenanthroline-reacting iron reacted directly with o-phenanthroline ("directly-reacting iron"), but the other half only reacted after addition of dithionite ("dithionite-reducible iron"). Directly-reacting iron appeared to be ferric iron which was located in the hydrophobic region of the particulate fraction. This ferric iron could be reduced by sulfhydryl groups of the protein, The dithionite-reducible iron was probably located at the surface of the particulate fraction and could not be reduced by sulfhydryl groups. Part of the dithionite-reducible iron was reduced by NADH or succinate. This substrate-reducible iron, probably less than 10% of the total iron, was located in the cytochrome b(1) region of the respiratory chain. It was not associated with SDH. The effect of ultraviolet irradiation, inhibitors and extraction of ubiquinone on the activities of SDH and succinate oxidase was examined. From these experiments, and those outlined above, a scheme for the sequence of the succinate oxidase chain of E. coli is proposed.

Text

2011-04-28

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http://hdl.handle.net/2429/34117

Biochemistry and Molecular Biology

University of British Columbia

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