Analyzing the functions of positions P204, R210, and E219 in a switched double mutant R210Q/Q252R in subunit a from Escherichia coli ATP synthase

The ATP synthase is a bipartite enzyme found in most living organisms and is located in the mitochondrial inner membrane of eukaryotes, thylakoid membranes of plants, and the plasma membrane of bacteria. The enzyme is composed of two parts, the membrane embedded F0 portion and the F1 hydrophilic sector. The membranous F0 subunits form the proton pathway. In bacteria the flow of protons from the periplasm into the cytoplasm is used to drive rotation of the central stalk within F1. The rotation of the stalk thus drives the synthesis of ATP in a 3 step process at the respective catalytic beta sites.;This study used the bacterium Escherichia coli as a source of the enzyme. In E. coli, F0 consists of ab2c10. The work in this study examined a previously described double mutant of the E. coli ATP synthase in which the strictly conserved amino acid of subunit alpha, R210, was switched with another conserved residue Q252. R210 is found on the fourth transmembrane helix and Q252 is found at a similar depth on the fifth transmembrane helix within subunit alpha. Since R210 is critical for the transfer of protons from the alpha to c subunit during rotation, it was surprising then, when the switched mutant still showed function because this mutation involved the removal of a highly conserved essential amino acid. In our work, further mutagenesis was applied to answer three questions about the function of this double mutant. First, since a spontaneous P204T mutation in the double mutant background showed enhanced function, we asked what role threonine has in protein function or proton translocation. Results indicated that mutation to alanine or serine showed similar enhancement, and therefore it appeared that removal of proline at this position was the key change.;Second, the role of glutamine in the "switched" double mutant (R210Q/Q252R) was examined by saturation mutagenesis at this position. This analysis included the examination of many mutant constructs representing all groups of the amino acids. Of the mutants, two became of interest because they were able to grow on the succinate minimal medium plates. These mutations are the R210G and R210I mutants. Results showed that replacement of glutamine by glycine at position 210 enhanced function. Therefore it appears that the glutamine plays no role in function.;Finally we examined whether protons follow the normal pathway in the "switched" triple mutant during proton translocation. It is known that in the wild type E. coli alpha subunit, alphaE219 is within the proton pathway and when mutated to Lysine the mutant retains function, but when mutated to glutamine, it does not. Results show that when these mutations were constructed in the "switched" mutant background, the same pattern held. Therefore it appears that protons may follow through the same wild type pathway, at least from the periplasm to the c subunit, in the "switched" mutant. The results do not suggest that the pathway mechanisms are different.