PROTON-TRANSLOCATING CARBOXYL OF SUBUNIT-C OF F1FO H-ATP SYNTHASE - THE UNIQUE ENVIRONMENT SUGGESTED BY THE PK(A) DETERMINED BY H-1-NMR()

Subunit c of the H+-transporting F1F0 ATP synthase (EC 3.6.1.34) is th ought to fold across the membrane as a hairpin of two or helices with a conserved Asp/Glu residue, centered in the second membrane-spanning helix, which is thought to function in H+ translocation. NMR studies i ndicate that the purified subunit c from Escherichia coli is also fold ed as a hairpin in a chloroform/methanol/H2O (4:4:1) solvent mixture [ Girvin, M. E., & Fillingame, R. H. (1993) Biochemistry 32, 12167-12177 ] and that the conserved Asp remains uniquely reactive in this solvent mixture [Girvin, M. E., Br Fillingame, R. H. (1994) Biochemistry 33, 665-674]. The pK(a) of Asp61 is of interest because of its unique reac tivity and because it is thought to protonate and deprotonate during e ach proton translocation cycle. We have determined the pK(a) value of the carboxyl group of the functional Asp in wild type and two function al, mutant subunit c proteins, i.e. the Ala24-->Asp (D24D61) and the A la24-->Asp/Asp61-->Asn (D24N61) mutant proteins. The pK(a) values were determined by H-1 NMR spectroscopy by measuring changes in the alpha and beta proton chemical shifts by constant time two-dimensional (2D) correlated spectroscopy. The pK(a) of Asp61 in the purified wild type protein was 7.1. This pK(a) was significantly higher than the pK(a) of the other two Asp residues, i.e. Asp7 and Asp44 which were 5.4 and 5. 6, respectively. The pK(a) of the two Glu residues in the protein were determined by 2D total correlation spectroscopy and found to be appro ximately 5.5. In the D24D61 mutant protein, the pK(a) values of both A sp24 and Asp61 were abnormally high, i.e. 7.1 and 6.9, respectively. I n the D24N61 mutant protein, where Asp24 functions in proton transloca tion, the pK(a) of the functional Asp24 was 6.9. These observations su ggest that the protein folds such that the local environment around As p61 (or Asp24) is more hydrophobic than in the general environment of the organic solvent. The changes in chemical shift of sequentially ass igned protons of other residues were followed as a function of pH as a general measure of structural changes in the protein. The analysis in dicates a global conformational change in the protein coincident with the deprotonation of Asp61. The most pronounced effect of deprotonatio n is seen in the polar loop region and in the N-terminus. The conforma tional changes in this region of the protein, which are coupled to pro tonation/deprotonation of Asp61, may mimic those taking place during A TPase-coupled proton translocation by the F1F0 complex.