A time lag in the onset of ATP-Pi exchange catalyzed by purified ATP-synthase (CF0-CF1) proteoliposomes and by chloroplasts.

The time course of ATP-Pi exchange which is catalyzed by the isolated chloroplast ATP synthase in phospholipid vesicles was studied. The following observations were made. (i) The onset of 32Pi incorporation into ATP lags behind ATP hydrolysis. The lag lasts for about 2 min at 37 degrees C and is followed by a steady-state rate which is constant for more than 30 min. Under the same experimental conditions, ATP hydrolysis shows an initial burst followed by a constant, slower rate. (ii) The initial lag is independent of Mg-ATP concentration in the range 0.2-5 mM and of the presence of ADP. In contrast, the steady-state rate of ATP-Pi exchange has an apparent Km of 0.3 mM for Mg-ATP and is stimulated by ADP. (iii) Increasing the temperature from 30 to 45 degrees C decreases the lag from 6 min to zero. The steady-state rate of ATP-Pi exchange is affected to a much smaller extent by the temperature in this range. (iv) The lag is insensitive to valinomycin or tetraphenylboron, while the steady-state rate is partially inhibited. Nigericin and protonophores affect both the lag and steady-state rate. (v) ATP-induced membrane potential formation, as followed by oxonol VI, does not correlate with the lag in its kinetics and temperature dependence. ATP-induced pH gradient formation could not be detected in the proteoliposome system. (vi) Light-triggered ATP-Pi exchange in chloroplasts shows essentially the same time course as the proteoliposome system, but the lag lasts for only about 20 s at room temperature and is unaffected by a preexisting proton gradient. These results suggest that the initial lag in ATP-Pi exchange does not reflect the time required for the buildup of a protomotive force (delta - mu H+) nor the time required to produce ADP. It is suggested, therefore, that the lag reflects an internal autocatalytic conformational change in the ATP-synthase complex which is initiated by ATP hydrolysis and which converts the enzyme from an "exclusive ATPase state" to a "reversible ATP-synthase state". This slow transition is not directly coupled to a trans-membrane pH or potential gradient.