Studies on the nature of the water-oxidizing enzyme: II. On the functional connection between the reaction-center complex and the water-oxidizing enzyme system Y in Photosystem II

Abstract

Laser-flash-induced absorption changes have been measured with a time resolution of 1–2 μs in PS II membrane fragments. The following was found. (1) In the presence of 0.5 mM K 3 [Fe(CN) 6] at pH = 6.0 absorption changes at 830 nm induced by repetitive flash excitation in PS II membrane fragments pretreated (10 min) with trypsin at pH = 7.5 are dominated by 10–15 μs kinetics. Under the same conditions the relaxation kinetics at 325 nm are slower by at least two orders of magnitude. (2) In the same samples μs components contributing to the overall kinetics of transient ultraviolet absorption changes have the same half-life time as the 830 nm relaxation kinetics within the range of 4.0 ≤ pH ≤ 8.0. (3) At pH = 6.0 the extent of 10–15 μs kinetics of ultraviolet absorption changes as a function of wavelength is characterized by peaks at 260 nm, 300 nm, 360 nm (positive) and 340 nm (negative). It is vanishingly small in the range of 315–325 nm. (4) In the presence of 1 mM hydroxylamine dark-adapted samples illuminated with a few flashes (n ≤ 8) revealed the same pattern at 270 nm and 830 nm as PS II membrane fragments trypsinized at pH = 7.5. In contrast to that, after preillumination with 70 flashes the absorption changes at 270 nm and 830 nm exhibit slower kinetics in the microsecond range with half-life times of 200–300 μs. These results are interpreted as a light requiring disconnection of Z from P-680 by hydroxylamine. (5) After subtraction of the difference spectrum for the reduction of the primary plastoquinone acceptor Q A the extent of the separated initial amplitudes (limited by time resolution of 1–2 μs) as a function of wavelength exhibit characteristic features, depending on the functional integrity of the water-oxidizing enzyme system Y. If the samples were deprived of their oxygen-evolving capacity the data obtained resemble the difference spectrum reported for oxidation of chlorophyll a in solution (Borg, D.C., Fajer, J., Felton, R.H. and Dolphin, D. (1970) Proc. Natl. Acad. Sci. USA 67, 813–820). On the other hand, a markedly different spectrum is obtained in samples with a functionally competent water-oxidizing enzyme system Y. Based on kinetical arguments this difference spectrum is inferred to reflect Z-oxidation in oxygen-evolving PS II membrane fragments. The shape of the Z ox Z difference spectrum appears to be slightly affected by the functional integrity of systems Y. (6) No direct evidence was obtained for the existence of a kinetically and spectrally distinguishable redox component D x between Z and P-680 in samples that were deprived of their oxygen-evolving capacity. On the basis of the present data the mode of functional coupling between system Y and P-680 is discussed. Furthermore, implications of possible changes in the electrostatic interactions between the redox active chromophors and the surrounding protein matrices are considered.