The protonization pattern of the endogenous donor component D1 which feeds electrons directly into chl-a + II has been analyzed in Tris-washed inside-out thylakoids with the aid of appropriate pH-indicators. It was found that under repetitive flash excitation the amount of protons released is proportional to the extent of D1-oxidation, depending on the time between the flashes. The kinetics of D1-oxidation (being practically the same as in normal Tris-washed chloroplasts) are faster than the proton release by two orders of magnitude. The results lead to the conclusion that D1 is protonized in the reduced state with pK(Dox 1) < 5 and becomes deprotonized in the oxidized state with pK(Dred 1) ≳ 8. The proton release is kinetically limited by a transport barrier. Implications on the interpretation of the proton release pattern in preparation with intact water oxidation are discussed.
