Isolation and functional aspects of the fumarate reductase involved in the phosphorylative electron transport of Vibrio succinogenes.
1. The fumarate reductase of Vibrio succinogenes, the terminal component of the electron transport chain of the bacterium, was extracted from the cytoplasmic membrane with Triton X=100 and purified to homogeneity (approx. 30-fold) by means of chromatograhy on hydroxyapatite and DEAE-Sephadex. The enzyme eluted from the ion-exchange column in two forms, one containing and the other lacking cytochrome b. 2. The enzyme lacking cytochrome b consisted of two peptides (Mr 79 000 and 31 000) which were present in a molar ratio of 1:1. The cytochrome-containing species contained an additional peptide (Mr 25 000) which was present in twice the molar amount of the others (2:1:1). 3. The hydrodynamic properties indicate that the functional enzymes consist of only one set of the corresponding peptides. 4. Each of the two enzyme molecules contains one protein-bound FAD which is linked to the Mr 79 000 peptide. Both enzyme species contain 9-10 mol iron-sulfur per mol of FAD which is associated with the Mr 79 000 and the Mr 31 000 peptide. Cytochrome b is present in an amount of 2 mol/mol of FAD, half of the cytochrome b has a midpoint potential of -20 mV. 5. The enzyme catalyzed two types of reaction. (a) Fumarate reduction by viologen radicals or anthrahydroquinonesulfonate, as well as succinate oxidation by ferricyanide or methylene blue, was independent of cytochrome b. (b) The activities of fumarate reduction by naphthohydroquinones and those of succinate oxidation by naphthoquinones were cytochrome b-dependent. This indicates that the electron transport from menaquinone to fumarate reductase in the membrane of the bacterium is mediated by a single component, cytochrome b (-20 mV). 6. The Km for fumarate was 0.35 mM with menadiol as the donor and that for succinate as 20 mM in the reverse reaction. Succinate oxidation was competitively inhibited by fumarate with a Ki of 0.35 mM.
D002462:Cell Membrane / Q000201:enzymology; D002852:Chromatography, Ion Exchange; D003580:Cytochromes / Q000032:analysis; D004579:Electron Transport; D004792:Enzyme Precursors / Q000032:analysis; D005650:Fumarates / Q000302:isolation & purification; D006859:Hydrogen / Q000302:isolation & purification; D006864:Hydrogenase; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D010088:Oxidoreductases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D013385:Succinate Dehydrogenase; D014733:Vibrio / Q000378:metabolism*
D003580:Cytochromes; D004792:Enzyme Precursors; D005650:Fumarates; D009097:Multienzyme Complexes; D006859:Hydrogen; C023310:H(2) fumarate reductase; D010088:Oxidoreductases; D006864:Hydrogenase; D013385:Succinate Dehydrogenase