Activation and Inactivation of CF0F1 Reconstituted in Liposomes Monitored by ATP Binding under Uni-Site Conditions

Abstract

The H+-ATPase from chloroplasts, CF0F1, is localized in thylakoid membranes where it catalyzes ATP synthesis and ATP hydrolysis coupled to a transmembrane proton transport. The activity of CF0F1 is strongly regulated. The enzyme exists in at least four different states: it can be oxidizedor reduced and in both redox states it can be either inactive E i ox , E i red or active Er a ox , E a red (1). Under physiological conditions, when the thylakoid membrane is energized CF0F1 is presumably in the reduced active state. Conversely, when the membrane is deenergized, CF0F1 is in the stable inactive state. It is transformed into its metastable active state when a transmembrane pH-difference, ΔpH and/or an electric potential difference, Δφ, is generated (2). In addition to the activation of CF0F1, the ATP synthesis is driven by ΔpH and Δφ. Therefore, when ATP synthesis is measured starting with CF0F1 in its inactive state, the enzyme has to be transferred into its active state before ATP synthesis takes place. In order to measure the kinetics of either activation or ATP synthesis both processes must be separated. In our previous work with thylakoid membranes this was achieved by preilluminating the membranes. Subsequently, an acid-base transition was carried out in a quenched flow apparatus and the rate of ATP synthesis was measured with the active enzyme (3). Similar experiments with CF0F1 reconstituted into liposomes require two acid-base transitions: in the first one CF0F1 is activated and in the second one ATP synthesis is measured. In this work we investigate the activation of CF0F1 reconstituted in reverse phase liposomes as a function of ΔpH generated by acid-base transitions and measure the stability of the active state.