EFFECT OF THE gammaM23-K MUTATION ON ATP
SYNTHASE ACTIVATION BY THE PROTONMOTIVE FORCE
AND ON COUPLING ATP HYDROLYSIS TO PROTON
TRANSLOCATION
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Abstract
The single-site mutation M23-K in the gamma-subunit of the E.coli ATP synthase has been
reported to perturb the energetic coupling between F1 and FO and to increase the transition state
activation energy for ATP hydrolysis (1). We have introduced the homologous mutation gamma-
M23-K in the ATP synthase of Rhodobacter capsulatus in order to study its phenotype by taking
advantage of the photosynthetic system.
In our hands, the most striking phenotypic difference to the wild-type complex was found in the
ATP hydrolysis activation by the protonmotive force. The higher ATP hydrolysis rate is best
observed when the activating protonmotive force is dissipated by addition of uncouplers. At low or
zero protonmotive force, though, this activated state decays and can be measured only for a limited
time. We have found that in the mutated enzyme, the half-life time of the light-activated state,
measured under the above conditions, is reduced from 13 to 4 s. Moreover, after a burst of ATP
synthesis triggered by a train of flashes, while the wild-type rapidly hydrolyzes the newly
synthesized ATP, the mutant does not, indicating that stabilization of the inactive state is even
higher at low ATP concentration.
The efficiency of proton coupling could be modified, both in the wild type (2) and mutated
strain according to the experimental conditions. While a large difference in efficiency could be
detected in the M23K mutated strain as compared to the wild type under certain conditions, in other
cases the difference was much more limited. A detailed analysis of the two phenotypes will be
presented and discussed in relation to the modulation of proton pumping activity and the possible
differences produced by the mutation.
(1) Al-Shawi MK, Ketchum CJ, Nakamoto RK.. (1997) J. Biol. Chem. 272: 2300-6.
(2) Turina P., Giovannini D., Gubellini F. and Melandri B.A. (2004) Biochemistry 43: 11126-34