Studies on the proton transport at system II in trypsin-treated spinach chloroplasts

The proton transport coupled with the DCMU-insensitive oxygen evolution mediated by K3[Fe(CN)6] in trypsin-treated chloroplasts (Renger, G. (1976) FEBS Lett. 69, 225--230) has been investigated with the aid of the pH indicator bromcresol purple. It was found that (1) the proton uptake from the outer aqueous phase observed in normal chloroplasts is completely suppressed by mild trypsin treatment; (2) a rather slow proton release into the external phase is detected which is insensitive to DCMU; (3) in the presence of DCMU, the extent of the proton release depends on the incubation time with trypsin in a similar manner as the average oxygen yield per flash. The results are interpreted by the assumption, that: (i) the reduced primary electron acceptor of System II, X 320-, does not become protonated, and (ii) the external acidification is caused by a passive efflux of protons, which are released by the watersplitting enzyme system Y into the inner phase of the thylakoids. The pK value of X 320- in trypsinated chloroplasts is estimated to be below 4.5. A possible pK shift caused by a modification of the proteinaceous barrier, which earlier (Renger, G. (1976) Biochim. Biophys. Acta 440, 287--300) was postulated to cover up the primary electron acceptor X 320, is discussed. Furthermore, the watersplitting enzyme system Y is inferred to be sensitive to deletereous attack from the outer aqueous phase mainly by secondary structural effects. Trypsination does not change the direction of the proton release in system Y.