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 Ravin, and no iron-sulfur clusters. Upon isolation, NDH-II loses activity, which can, nevertheless, be restored by incubation with phuspholipids. 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 K-m, for NADH was found to be similar to6 muM, 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 aa(3) 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.