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Title: Enzymology and energetics of thiosulphate oxidation by Thiobacillus A2
Author: Lu, Wei-ping
ISNI:       0000 0001 3565 378X
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1983
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The mechanism of thiosulphate oxidation by Thiobacillus versutus (A2) was investigated in an attempt to elucidate the enzyme system and the energetic processes involved. The theoretical maximum growth yield of the organism on thiosulphate was 7 (g dry wt/mol). Thiosulphate oxidation was coupled to the electron transport chain through c-type cytochrome(s). Cell-free extracts catalyzed the complete oxidation of thiosulphate and linked the associated electron transport to ATP synthesis with a P/0 ratio of about one. On the basis of these data and a knowledge of the biochemistry of the organism, a requirement reduction of one mole of approximately three ATP for NAD+ was deduced and the involvement of an oxygenase in thiosulphate oxidation ruled out. Enzymes for conversion of thiosulphate to sulphate were located in the cytoplasm, but oxygen uptake required the membrane fraction, which provided cytochrome c and cytochrome oxidase and could be replaced by its counterparts from mammalian sources. The crude extract was fractionated by ammonium sulphate, then resolved into three major fractions involved in thiosulphate oxidation by chromatography on DEAE-Sepharose-CL 6B. Four essential components, enzyme A, enzyme B, cytochrome c_511 and cytochrome c_512.5 and sulphite : cytochrome c oxidoreductase were finally highly purified from the three fractions by means of gel filtration, hydrophobic interaction chromatography, isoelectric focusing, chromatofocusing and gel electrophoresis. Enzyme A (Mr 16,000) and Enzyme B (Ilr 64,000) were colourless proteins both necessary for the thiosulphate-oxidizing reaction. Cytochrome c_511 (Mr 260,000) and cytochrome c_512.5 (Mr 56,000) were multihaem c-type cytochromes, each with a dual Em, 7 of -115, +240 and about -50 and *220 mV respectively. The four components, each comprising 0.6 to 1.57. of the cellular protein, catalyzed complete oxidation of thiosulphate in the presence of an electron transport system. The reaction proceeded in an integral way as neither cleavage of thiosulphate nor any intermediates (e.g. sulphite) were detected and the individual or specific functions of the four components were not identified. The two cytochromes may serve not only as electron carriers but could also have other enzyme functions. Sulphite : cytochromo c oxidoreductase was an important component in the enzyme system, but further study of its function was hampered by its low concentration (less than 0.52 of the cellular proteins) and its intimate association with cytochrome c_511. Rhodanese was proven not to be a part of the thiosulphate-oxidizing system. A generalized mechanism and a model system are proposed for the path of thiosulphate oxidation to sulphate, the involvement of the four essential components and the role of dual Em 7 of the cytochromes in feeding electrons into ATP and NADII-generating electron transport. The problems remaining to be resolved and possible routes to their solution, are outlined.
Supervisor: Not available Sponsor: Committee of Vice-Chancellors and Principals of the Universities of the United Kingdom ; Jiao yu bu, China
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry ; QP Physiology