(Received for publication, July 7, 1995; and in revised form, September 13, 1995)
From the
The substitution of arginine at position 210 in the a subunit of Escherichia coli FF
-ATPase by either lysine or alanine
causes dominance in complementation tests with a chromosomal c subunit mutation. Reversal of dominance was achieved for the a R210K mutation but not for the a R210A mutation by the
presence of an aspartic acid residue at position 50 or at position 252
in the a subunit. It was concluded that position 210 in
putative helix 4 of a previously proposed model of the a subunit is close to position 252 in putative helix 5 and to
position 50 in putative helix 1. The juxtaposition of residues 252 and
210 was also indicated by the observation that the double mutant a R210Q/Q252R was partially functional. A revertant of the partially
functional double mutant, isolated on succinate medium, was found to
contain a third mutation resulting in Pro-204 in the a subunit
being replaced by threonine. That the revertant phenotype was due to
the a P204T change was confirmed by site-directed mutagenesis.
ATP synthesis in the revertant strain was at near normal levels as
judged by growth yield experiments, but the revertant strain was unable
to pump protons in response to ATP hydrolysis.
The FF
-ATPase enzyme complex catalyses
the terminal step in oxidative phosphorylation and photophosphorylation
and is located in mitochondrial, chloroplast, and bacterial membranes.
In Escherichia coli, the enzyme comprises eight nonidentical
subunits, a, b, c,
,
,
,
, and
, encoded by the genes uncB, F, E, A, D, G, H, and C, respectively(1) . The a, b, and c subunits are integral membrane proteins and form the F
portion of the complex, which can function as a proton pore. The
,
,
,
, and
subunits are peripheral membrane
proteins forming the F
-ATPase portion of the complex, which
retains ATP hydrolytic activity when removed from the membrane. The a, b, and c subunits of the proton pore are present
in a stoichiometry of 1:2:6-12(2) , and all are required
for proton translocation. Residues essential for proton translocation
have been found in the a and c subunits(2) .
It has been proposed that the proton pore involves four amino acids:
Arg-210, Glu-219, and His-245 on the a subunit and Asp-61 on
the c subunit(3) . Of these, only Arg-210 is
absolutely conserved, but all c subunits contain an acidic
residue equivalent to Asp-61. In E. coli, neither Arg-210 nor
Asp-61 can be substituted by other residues without complete loss of
proton-coupled ATP
synthesis(4, 5, 6, 7, 8, 9) .
The remaining two residues required for proton translocation are not
strictly conserved, and some amino acid substitutions at either
position retain some proton translocating
activity(4, 5, 6, 10, 11) .
In order to understand the mechanism of proton translocation, information on the positions of these essential residues is required. There is a considerable body of evidence indicating that the c subunit forms a helical hairpin structure, placing Asp-61 in the center of the membrane (for review, see (12) ). However, the structure of the a subunit is less clear, with a number of different models being proposed(2, 13, 14, 15, 16, 17) . These models differ in the positioning, within the membrane, of the amino acid residues essential for proton translocation.
The use of site-directed mutagenesis in combination with analysis of second site revertants has proved a powerful tool in investigations of structure and function. For example, the finding that the essential aspartic acid residue in the c subunit can be moved to position 24 on the other helix and still retain some activity provides confirmation of the hairpin structure of the c subunit(18) . We have previously identified amino acid changes in second-site mutants that suppress dominance of the a subunit mutant in which Arg-210 is replaced by lysine(14) . Analysis of these revertants suggested that position 50 on putative helix 1 was adjacent to Arg-210 on helix 4 in the five-transmembrane helix model of the a subunit(14) . In the present study, we have extended this work to show that Arg-210 is also close to position 252 on putative helix 5. Furthermore, the residues at positions 210 and 252 can be exchanged with partial retention of function.
Figure 1: Helix diagrams of a subunit putative helices 3 and 5. Boxed numbers represent growth yields of the various mutant strains relative to a wild-type value of 100 and an uncoupled value of 0. Complete reversal of dominance would be indicated by a value of 100. See text.
The R210A mutation was similar to the R210K mutation in that it was found to be dominant in complementation tests with a chromosomal c subunit mutant. If the reversal of dominance by aspartate at positions 50, 163, and 252 in the R210K mutant was due to salt bridge formation with lysine, then reversal of dominance should not be achieved in the R210A mutant. Reversal of dominance did not occur in the R210A/V50D or the R210A/Q252D double mutants, but it did occur in the R210A/F163D double mutant (Table 2). The F163D mutation is therefore exerting its effect directly. These results suggest that position 210 is close to positions 252 and 50 but not to position 163. Helix 4 is therefore oriented in such a way that Arg-210 is close to helix 5 rather than to helix 3. Such an orientation would also mean that Glu-219 and His-245 do not directly interact(10) .
Figure 2: Growth of mutant strains on succinate minimal medium after 2 days at 37 °C. The high rate of reversion of these strains meant that an accurate growth rate in liquid medium could not be obtained. 1, Q252R; 2, R210Q; 3, R210Q/Q252R; 4, R210Q/Q252R/P204T.
Membranes were prepared from each of the three
strains and assayed for ATPase activity and atebrin
fluorescence-quenching activity. The ATPase activities indicated that
there was a somewhat reduced assembly in each of the mutant strains,
with the R210Q/Q252R double mutant the most severely affected (Table 3). This double mutant also gave the largest inhibition of
ATPase activity when F was bound to the membranes (see Table 3). ATP-dependent fluorescence quenching was absent in the
R210Q mutant, as found previously(4) , and was also essentially
absent in the R210Q/Q252R double mutant (Table 3). The Q252R
mutant showed a low ATP-dependent atebrin fluorescence quenching
activity (Table 3).
The arginine residue at position 210 of the a subunit of the E. coli ATP synthase is absolutely conserved and is essential for proton-coupled ATP synthesis. Previously, evidence had been obtained that position 210 was close to position 50 in the folded a subunit structure(14) . The work described here leads to the conclusion that position 210 is also close to position 252. If this is the case, then it would preclude any direct interaction between His-245 and Glu-219 (10) but would be consistent with a close interaction between Gly-218 and His-245 (32) (see Fig. 3). The evidence for the latter interaction would appear to be stronger in that the double mutant G218K/H245G is essentially wild-type(32) , whereas the double mutant E219H/H245E is only marginally different in energy coupling from the H245E mutant(10) . Furthermore the E219H mutant can be changed from essentially uncoupled to fully coupled by a second mutation R140H(33) , which is also consistent with this face of helix 4 being close to helix 3 rather than helix 5. The positions of these residues in a five-helix model of the a subunit are shown in Fig. 3. Arg-210 is placed close to position 50 in helix 1 and position 252 in helix 5 and is also able to interact with Asp-61 in the c subunit. The other two residues required for proton translocation, Glu-219 and His-245 of the a subunit, are also placed where they can interact with the c subunit Asp-61. The a subunit helices are positioned such that Arg-210 and Glu-219 are able to interact with the Asp-61 of different c subunits.
Figure 3:
A, proposed transmembrane helices of the a and c subunits from E. coli F-ATPase. B, proposed packing of the
transmembrane helices of the a, b, and c subunits of the E. coli F
-ATPase. Amino acid
residues discussed in the text are
indicated.
Work described by Vik and Antonio (34) is difficult to rationalize with this model in that they have concluded that position 252 is close to 219. They found that the double mutants Q252E/E219G and Q252E/E219K were functional. However Q252E alone is also functional so it might be that lysine or glycine at position 219 can functionally replace glutamate or allow other residues to take over that role even when the wild-type residue glutamine is at position 252. The other difference between the data reported by Vik and Antonio (34) and that reported in the present work is the effect of the replacement of Gln-252 by arginine. Their finding that the Q252R mutant was unable to grow on succinate may be due to different background strains used.
The retention of function after switching residues 210 and 252, albeit at a reduced level, is strong evidence for the juxtaposition of Arg-210 and Gln-252. The result is directly analogous to that obtained by Miller et al.(18) when they demonstrated retention of function in a mutant in which the essential aspartate at position 61 in the c subunit was shifted to position 24. A further similarity between the Asp-61 shift in the c subunit and the Arg-210 shift in the a subunit is the ability to improve function by additional mutations in helix 4 of the a subunit (35) . The major difference between the Asp-61 shift and the Arg-210 shift is the observation that in the latter case ATP-dependent proton pumping is lost, whereas ATP synthesis is retained. In the Asp-61 shift experiments, both of these activities were retained(18) . This difference may be rationalized by a consideration of the different positions the c subunit Asp-61 and the Arg-210 of the a subunit occupy in the proton-translocating pathway. It would appear that the c subunit Asp-61 is located at about the center of the bilayer (12) and is intimately involved in the movement of protons in both directions. Evidence has been obtained that a subunit residues Glu-219 and His-245 are also involved in proton translocation, and it has been proposed that Arg-210 interacts with the c subunit Asp-61 from the opposite side of the bilayer to the residues Glu-219 and His-245(14) . The results reported in the present work would indicate that Arg-210 of the a subunit is placed between the cytoplasm and the c subunit Asp-61. Thus Arg-210 would normally receive protons for transfer to Asp-61 during ATP hydrolysis, but the shift of the arginine residue has disrupted the pathway, presumably within the a subunit, by which the proton reaches Arg-210 from the cytoplasmic side of the membrane. This disruption would not affect ATP synthesis since the movement of protons from the periplasm to the c subunit Asp-61 via His-245 and Glu-219 of the a subunit would proceed normally and arginine at position 252 is still able to interact with Asp-61 of the c subunit. Given that there are 6-12 c subunits and one a subunit and that the Asp-61 in each c subunit is required for activity(36) , movement of the a subunit with respect to the c subunits is also required for activity. If the movement is a rotation of the a subunit within a ring of c subunits(13) , then it is reasonable that, despite the considerable shift in the position of the key arginine residue, some function could be retained provided that the new position is in the same plane of the membrane. The same argument would apply to the shift in the aspartate residue in the c subunit from position 61 to position 24(18) . The concept of rotation now forms the basis of many models for the mechanism of the ATP synthase.(34, 37, 38, 39, 40, 41) .