Effects of trypsin and bivalent cations on P-680+-reduction, fluorescence induction and oxygen evolution in Photosystem II membrane fragments from spinach

Abstract

Laser-flash-induced absorption changes at 830 nm, fluorescence-induction curves and the average oxygen yield per flash have been measured in spinach Photosystem II membrane fragments as a function of trypsin treatment and its modification by CaCl2. The following was found. (i) The relative contribution of the nanosecond relaxation to the overall decay kinetics of 830 nm absorption changes reflecting the P-680+-reduction decreases as a function of incubation time with trypsin. Simultaneously, mild treatment at pH = 6.0 markedly increases the extent of 200 μs kinetics that highly revert back to nanosecond kinetics by CaCl2 addition. After harsher trypsin treatment (pH = 7.5) pH-dependent 2–20 μs kinetics appear that cannot be reverted to nanosecond kinetics by CaCl2. (ii) The CaCl2-induced restoration of nanosecond kinetics is mainly due to a Ca2+-induced effect rather than to a functional role of Cl−. Sr2+ can substantially substitute for Ca2+, whereas Mg2+, Mn2+ and monovalent ions are almost inefficient. (iii) A quantitative correlation between the extent of the nanosecond kinetics and the average oxygen yield per flash was not observed. (iv) If CaCl2 is present in the assay medium for trypsin treatment the samples are markedly protected to proteolytic degradation. This effect mainly refers to the reaction pattern of the acceptor side. Other bivalent cations can substitute Ca2+ for its protective function. (v) The CaCl2-induced protection to proteolytic attack is extremely sensitive to a very short trypsin pretreatment that does hardly affect the shape of the fluorescence induction curve. The results are discussed in relation to the functional and structural organization of Photosystem II.