Site-directed mutagenesis of two conserved charged amino acids in the N-terminal region of alpha subunit of E. coli-F(0)F(1).

Two conserved charged amino acids of the N-terminal 'crown' region of the alpha subunit of E. coli-F(1), alpha-D36 and alpha-R40 were exchanged for chemically related (alpha-D36-->E, alpha-R40-->K) or unrelated amino acids (alpha D-36-->K, alpha R40-->G), respectively, by employing oligonucleotide-directed mutagenesis. ATP formation and ATP hydrolyzing activity of isolated plasma membrane vesicles was strongly inhibited in mutant HS2 (alpha-D36-->K), but only slightly affected in the other mutants. The inhibition is not due to a lower content of F0F1 in HS2. In this mutant the extent of the proton gradient generated by ATP hydrolysis was more than 80% inhibited; in all other transformants much smaller effects were observed. The proton gradient established by NADH oxidation was 33% decreased in HS2, but was decreased to a lesser extent in all other mutants. After blockage of F0 by DCCD treatment, the same NADH-induced proton gradient was obtained in all transformants including HS2. This and the fact that the activity of NADH oxidation was unchanged indicate increased proton leakiness of F0F1 carrying the alpha-D36-->K mutation. In F1 alpha-D36 is located in a domain contacting the beta subunit in the vicinity of the arginine beta-R52. The effect of alpha-D36-->K replacement on catalysis and coupling thus may be due to an electrostatic repulsive effect in the crown region which alters the alpha and beta interaction.

Insertion of a "chloroplast-like" regulatory segment responsible for thiol modulation into gamma-subunit of F0F1-ATPase of the cyanobacterium Synechocystis 6803 by mutagenesis of atpC.

A regulatory sequence in the gamma subunit of the F0F1-ATPase complex of higher plant chloroplasts, responsible for so-called thiol modulation, is absent in the corresponding polypeptides of the cyanobacterial complexes analysed so far. We have modified the atpC gene encoding this gamma subunit in Synechocystis 6803 by site-directed mutagenesis. A segment was introduced coding for nine additional amino acids, including the two functional cysteines, which constitutes the sequence of the respective element in the chloroplast gamma subunit. The growth rate as well as the rate of photosynthesis of the transformant was comparable to that of the wild-type, but the transitory increase in respiration observed immediately after a period of illumination was significantly lower in the mutant than in the wild-type. The F1 subcomplex solubilized from thylakoid membranes of both the wild-type and the transformant can be activated by trypsin to yield Ca(2+)-dependent ATPase activity, but only the F1 from the transformant can be activated by the thiol reagent dithiothreitol.