A new type-II NADH dehydrogenase from the archaeon Acidianus ambivalens: characterization and in vitro reconstitution of the respiratory chain.
A new type-II NADH dehydrogenase (NDH-II) was isolated from the hyperthermoacidophilic archaeon Acidianus ambivalens. This enzyme is a monomer with an apparent molecular mass of 47 kDa, containing a covalently bound flavin, and no iron-sulfur clusters. Upon isolation, NDH-II loses activity, which can, nevertheless, be restored by incubation with phospholipids. Catalytically, it is a proficient NADH:caldariella quinone oxidoreductase (130 mmol NADH oxidized/mg protein(-1)/min(-1)) but it can also donate electrons to synthetic quinones, strongly suggesting its involvement in the respiratory chain. The apparent Km for NADH was found to be approximately 6 microM, both for the purified and membrane-integrated enzyme, thus showing that detergent solubilization and purification did not affect the substrate binding site. Further, it is the first example of a type-II NADH dehydrogenase that contains the flavin covalently attached, which may be related to the need to stabilize the otherwise labile cofactor in a thermophilic environment. A fully operative minimal version of Acidianus ambivalens respiratory system was successfully reconstituted into artificial liposomes, using three basic components isolated from the organism: the type-II NADH dehydrogenase, caldariella quinone, the organism-specific quinone, and the aa3 type quinol oxidase. This system, which mimics the in vivo chain, is efficiently energized by NADH, driving oxygen consumption by means of the terminal oxidase.
Gomes
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CM
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
Bandeiras
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Teixeira
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D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004579:Electron Transport; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009245:NADH Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010084:Oxidation-Reduction; D017386:Sequence Homology, Amino Acid; D016943:Sulfolobaceae / Q000201:enzymology* / Q000235:genetics
C088908:NADH dehydrogenase II; D009245:NADH Dehydrogenase