Studies on the nature of the water-oxidizing enzyme. I. The effect of trypsin on the system-II reaction pattern in inside-out thylakoids

Abstract

The effect of mild trypsination on the system-II reaction pattern of inside-out thylakoids has been analyzed by measurements of oxygen yield, fluorescence induction and laser-pulse-induced absorption changes at 320 and 830 nm. The following was found. (1) The average oxygen yield per flash drastically declines after trypsination at pH 7.4, while at pH 6.5 only small effects are observed. (2) The area over the fluorescence induction curves becomes reduced by 30–40% after trypsination at either pH 6.5 or 7.4, but in the latter case the maximum level is attained only after addition of hydroxylamine as PS-II donor. On the other hand, the area over the induction curve in the presence of DCMU, which is 10–12 times smaller than without DCMU, remains unaffected by trypsin treatment. (3) The oscillation pattern of the oxygen yield induced by a flash train in dark-adapted inside-out thylakoids is not markedly affected by trypsin treatment, even at more than 80% inhibition of the oxygen-evolving capacity. (4) After trypsination of inside-out thylakoids, a large 10 μs decay arises in the relaxation kinetics of the 830 nm absorption changes, whereas the 320 nm absorption changes are dominated by a rather slow decay. (5) The half-life time of the microsecond kinetics at 830 nm elicited by trypsination of inside-out thylakoids reveals almost the same pH dependence as the corresponding relaxation kinetics in Tris-washed inside-out thylakoids. (6) The relaxation kinetics of the absorption changes at 320 and 830 nm in Tris-washed inside-out thylakoids become significantly modified after trypsin treatment. Based upon these findings, it is concluded that beyond the well-characterized polypeptides with 16, 23 and 33 kDa there exists a further protein that is exposed to the inner side of the thylakoid and that affects the electron transport in system II. The nature and the physiological role of this polypeptide still remain to be elucidated.