The single-point mutation gammaM23-K introduced in the ATP synthase of E. coli has been reported to perturb the coupling efficiency between ATP hydrolysis and proton pumping as measured with the ACMA assay (1). Supporting this conclusion, the ATP synthesis rate was more affected compared to wild-type than the ATP hydrolysis rate, by about threefold (2). In addition, a study of interaction between subunits indicated that, in the mutated complex, the epsilon subunit inhibition of ATPase activity was not relieved upon binding of F1 to the membrane as observed in the wild-type (2). With the aim of further investigating the uncoupling process in a photosynthetic system in which analysis of the kinetics of the phosphorylating proton fluxes is possible (3), we have introduced this same mutation in the ATP synthase of Rb. capsulatus.In this organism, ATP synthesis and hydrolysis rates were impaired to a similar extent, both to approximately 1/3 of wild-type. Analysis of phosphorylating proton fluxes and associated ATP synthesis in the mutated and wild-type enzymes has not revealed uncoupling.However, the protonmotive force-activated state (measured as the transient increase of the ATP hydrolysis rate upon addition of uncouplers to energised vesicles), decayed extremely fast compared to wild-type. In agreement with this finding, the coupled proton flux through FoF1 induced by a single flash, which is usually observed in the wild-type enzyme in the presence of ADP and Pi, was completely absent. We conclude that the gammaM23-K mutated ATP synthase of Rb. capsulatus is an excellent system for studying the mechanism of ATP synthase activation by the protonmotive force. References [1] K. Shin, R.K. Nakamoto, M. Maeda, M. Futai, J. Biol Chem. 267 (1992) 20835–20839. [2] M.K. Al-Shawi, C.J. Ketchum, R.K. Nakamoto, J. Biol Chem. 272 (1997) 2300–2306. [3] B.A. Feniouk, D.A. Cherepanov, W. Junge, A.Y. Mulkidjanian, Biochim. Biophys. Acta 506 (2001) 189–203.
S. Anefors, D. Giovannini, P. Turina, W. Junge, A. Mulkidjanian, B. Feniouk, et al. (2004). A single-point mutation in the ATP synthase of Rb. capsulatus impairing the stability of the protonmotive forceactivated state.
A single-point mutation in the ATP synthase of Rb. capsulatus impairing the stability of the protonmotive forceactivated state
GIOVANNINI, DONATELLA;TURINA, MARIA PAOLA;MELANDRI, BRUNO ANDREA
2004
Abstract
The single-point mutation gammaM23-K introduced in the ATP synthase of E. coli has been reported to perturb the coupling efficiency between ATP hydrolysis and proton pumping as measured with the ACMA assay (1). Supporting this conclusion, the ATP synthesis rate was more affected compared to wild-type than the ATP hydrolysis rate, by about threefold (2). In addition, a study of interaction between subunits indicated that, in the mutated complex, the epsilon subunit inhibition of ATPase activity was not relieved upon binding of F1 to the membrane as observed in the wild-type (2). With the aim of further investigating the uncoupling process in a photosynthetic system in which analysis of the kinetics of the phosphorylating proton fluxes is possible (3), we have introduced this same mutation in the ATP synthase of Rb. capsulatus.In this organism, ATP synthesis and hydrolysis rates were impaired to a similar extent, both to approximately 1/3 of wild-type. Analysis of phosphorylating proton fluxes and associated ATP synthesis in the mutated and wild-type enzymes has not revealed uncoupling.However, the protonmotive force-activated state (measured as the transient increase of the ATP hydrolysis rate upon addition of uncouplers to energised vesicles), decayed extremely fast compared to wild-type. In agreement with this finding, the coupled proton flux through FoF1 induced by a single flash, which is usually observed in the wild-type enzyme in the presence of ADP and Pi, was completely absent. We conclude that the gammaM23-K mutated ATP synthase of Rb. capsulatus is an excellent system for studying the mechanism of ATP synthase activation by the protonmotive force. References [1] K. Shin, R.K. Nakamoto, M. Maeda, M. Futai, J. Biol Chem. 267 (1992) 20835–20839. [2] M.K. Al-Shawi, C.J. Ketchum, R.K. Nakamoto, J. Biol Chem. 272 (1997) 2300–2306. [3] B.A. Feniouk, D.A. Cherepanov, W. Junge, A.Y. Mulkidjanian, Biochim. Biophys. Acta 506 (2001) 189–203.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
