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.

EFFECT OF THE gammaM23-K MUTATION ON ATP SYNTHASE ACTIVATION BY THE PROTONMOTIVE FORCE AND ON COUPLING ATP HYDROLYSIS TO PROTON TRANSLOCATION

REBECCHI, ALBERTO;TURINA, MARIA PAOLA;MELANDRI, BRUNO ANDREA
2006

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.
14-th European Bioenergetic Conference 2006
319
319
Rebecchi A .; Feniouk B.; Turina P.; Melandri B.A.
File in questo prodotto:
Eventuali allegati, non sono esposti

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/123036
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? 0
social impact