Cooperative transient trapping of photosystem II protons by the integral membrane portion (CF0) of chloroplast ATP-synthase after mild extraction of the four-subunit catalytic part (CF1)

The ATP-synthase in chloroplasts is built from two blocks, CF0, which is integral to the thylakoid membrane and which serves as a proton channel, and CF1, attached to CF0, which is catalytically active. This study is aimed at understanding proton conduction through CF0. By a mild procedure we extracted <10% of total CF1, predominantly the four-subunit CF1 without the δ subunit. Extracted chloroplasts were excited with short flashes of light and the time course of the transmembrane potential and of the pH changes in both phases was measured spectrophotometrically. Mild extraction of CF1 caused two effects. (i ) Up to 50% of the protons rapidly released from water oxidation transiently escaped detection in the thylakoid interior. (ii ) The initial extent of the transmembrane potential was decreased by some 10% (20-μs resolution). Protons that were not detected inside appeared in the external phase after having passed the thylakoid membrane. pH titrations of the transient loss of protons produced an extremely sharp transition (near pH 7.5) as if six protons were buffered in a strictly cooperative manner. These effects were reversed upon addition ofN,N′ -dicyclohexylcarbodiimide, which, among other actions, blocks the proton channel through CF0. We interpret these observations as follows. (i ) CF0 incorporates proton binding groups, which can act in a hexacooperative way. These groups are located near the middle of the membrane. (ii ) After extraction of CF1, protons produced during water oxidation have very rapid access to these groups, but they pass the full span of the membrane more slowly: buffering precedes conduction through CF0.