ESSENTIAL ASPARTATE IN SUBUNIT-C OF F1F0 ATP SYNTHASE - EFFECT OF POSITION-61 SUBSTITUTIONS IN HELIX-2 ON FUNCTION OF ASP(24) IN HELIX-1

Subunit c of the F1F0 type, H+-transporting ATP synthase contains an e ssential Asp that is thought to function in H+ transport. Subunit c fo lds as a helical hairpin of two transmembrane helices with the essenti al Asp centered at residue 61 in transmembrane helix-2. Miller et al. (Miller, M. J., Olderburg, M., and Fillingame, R. H. (1990) Proc. Natl . Acad. Sci. U. S. A. 87, 4900-4904) have described a functional subun it c variant in which the essential Asp was moved from helix-2 to resi due 24 on helix-1 with replacement of Asp(61) by Gly. The function of the A24D/D61G subunit c variant is not optimal. In this study, 11 posi tion 61 variants of an A24D subunit c were generated by site-directed mutagenesis in order to test the importance of the position 61 residue . Three functional combinations were found with activities in the orde r: A24D/D61N > A24D/D61G greater than or equal to A24D/D61S. Other sub stitutions at position 61, including Ala and Cys, did not support func tion in the A24D protein. Although the A24D/D61N variant showed the hi ghest rates of ATPase-coupled H+ transport, its F-0 was inactive in pa ssive H+ transport when F-1 was stripped from the membrane. On the oth er hand, passive H+ transport by A24D/D61G and A24D/D61S stripped memb ranes approached that of wild type. The defect in function in these tw o mutants must be ascribed to events related to coupling ATPase and H transport. An A24D subunit c (with Asp at both position 24 and 61) wa s also generated. Its function proved to be pH-dependent. Activity app roaching that of wild type was observed at pH 7.0, but function was al most completely lost at pH 7.8. The pH dependent loss of ATP synthase function led to a slowing of growth on succinate as carbon source on r aising the pH from 7.0 to 7.8. In the A24D mutant, with a second Asp a t position 61, we postulate that 1 Asp must be protonated before the o ther can function in H+ transport.