RECONSTITUTION OF UBIQUINONE-LINKED FUNCTIONS IN MEMBRANE-VESICLES FROM A DOUBLE QUINONE MUTANT OF ESCHERICHIA-COLI

Membrane vesicles prepared from a mutant of Escherichia coli that is deficient in ubiquinone and menaquinone are markedly defective in their ability to oxidize d-lactate, NADH, and other substrates, and these electron donors do not drive proline transport effectively. Reconstitution of mutant vesicles with each of eight ubiquinone side-chain homologues (ubiquinone-1 through ubiquinone-8) under appropriate conditions restores certain activities to levels that are comparable to those of vesiclesprepared from the parent strain, while menaquinone homologues are ineffective. However, there are dramatic differences in the efficiency of reconstitution with various ubiquinone homologues. High levels of d-lactant oxidation and d-lactate-driven proline transport are restored with both long- and short-chain homologues, but ubiquinone-1 functions at much lower membrane-bound concentrations than ubiquinone-8 NADH is oxidized at a high rate by vesicles from the parent, but does not drive transport effectively, and addition of ubiquinone-1 stimulates proline transport over tenfold with no increase in NADH oxidation. In contrast, addition of ubiquinone-8 causes very little stimulation of proline transport. Moreover, in mutant vesicles, reconstitution with ubiquinone-1 results in both NADH oxidation and NADH-driven proline transport, while ubiquinone-8, on the other hand, reconstitutes NADH oxidation but has almost no effect on proline transport. The results indicate that functional reconstitution with ubiquinone side-chain homologues does not result simply from binding of the quinones to the membrane, but requires interaction of the bound quinone with specific sites. Moreover, the data suggest that ubiquinone-8 is not freely mobile within the membrane and imply that the enhanced mobility of ubiquinone-1 allows it to shunt reducing equivalents from NADH dehydrogenase to a segment of the respiratory chain containing the site at which the electrochemical proton gradient is generated. In addition, experiments with dithiothreitol as a reducing agent suggest that ubiquinone-8 is not accessible from the surface of the membrane. © 1979, American Chemical Society. All rights reserved.