Studies on the nature of the water-oxidizing enzyme: III. Spectral characterization of the intermediary redox states in the water-oxidizing enzyme system Y

Abstract

Spectral changes in the ultraviolet region (250–380 mn) that are caused by redox transitions within the water-oxidizing enzyme system Y were analyzed by measuring time-resolved (1–2 μs) absorption changes in dark-adapted PS II membrane fragments. In order to eliminate effects due to the interference with binary oscillation at the acceptor side the experiments were performed in trypsinized (at pH = 6.0) samples with 0.4 mM K 3[Fe(CN) 6] as exogenous oxidant. In the presence of 10 mM CaCl 2 PS II-membrane fragments trypsinized at pH = 6.0 are fully competent in oxygen evolution compared with the control (measured as average oxygen yield per flash). It was found: (1) Excitation of dark-adapted samples with a train of saturating laser flashes (7 ns full width at half maximum, FWHM) induces absorption changes with resolved kinetics in the micro- and millisecond range. Except for the kinetically unresolved initial amplitude the other transient compoents are strongly dependent (in extent and half-life time) on the flash number. (2) After elimination of the oxygen-evolving capacity by 1 mM hydroxylamine (NH 2OH) binary oscillation are completely lacking of absorption changes at 325 nm. The remarkably smaller absorption change in the first flash can be explained by partial oxidation of acceptor ‘C400’ due to K 3[Fe(CN) 6]. (3) A significantly different pattern of the absorption changes is observed if phenyl- p-benzoquinone is used instead of K 3[Fe(CN) 6]. Based on the oscillations of the initial amplitude of the absorption changes, exogenous quinones are assumed to give rise to a more complicated reaction pattern. (4) The spectral analysis performed on the basis of Kok's model (Kok, B., Forbush, B. and McGloin, P.M. (1970) Photochem. Photobiol. 11, 457–475) by the use of α, β and the apparent [ S 0] [ S 1] ratio experimentally determined via oxygen-yield measurement leads to the separation of the difference spectra Δε( S i → S i + 1 ) for the redox transitions in system Y. The obtained difference spectra are almost identical for the S 0 → S 1 and S 2 → S 3 transitions, while for S 1 → S 2 a markedly different spectrum has been observed. The deviations are especially pronounced in the range around 325 nm. (5) The relaxation kinetics of absorption changes at 325 nm that are characterized by a 1 ms half-life time oscillate in their amplitude synchronously with the oxygen yield in a flash train. The amplitude of these kinetics as a function of wavelength corrected for the Z ox Z difference spectrum closely resembles the calculated spectrum for the S 3 → S 0 transition. Based on the experimental data a model is proposed for the molecular mechanism of photosynthetic water oxidation that is an extension of our previous hypothesis (Renger, G. (1977) FEBS Lett. 81, 223–228). Accordingly, the catalytic site for water oxidation is assumed to be a binuclear manganese center which undergoes redox reactions at the manganese centers only during S 1 → S 2 and S 3 → S 0 transitions. The implications of this model are discussed.