The temprature dependence of the rate constants of the univalent redox steps Y,"'S, + Y,S,+, (i = 0.1,21 and Y.""S, 3 (Y&) + YSO + Oz in the water oxidase was investigated by measuring time resolved absorption changes at 355 nm induced by a laser flash train in dark adapted PS II membrane fragments from spinach. Activation energies of 5.0. 12.0 and 36.0 kJ/mol were obtained for the reactions Y,"*S, + YrSr+, with i = 0.1 and 2, respectively. The reaction Yp'S, + (Y&) 3 Y$, + 0: exhibits a temperature dependence with a characteristic break point at 279 K with activation energies of 2L1 kJ/mol (T > 279 K) and 46 kJ/mol (T < 279 K). Evaluation of the data within the framework of the classical Marcus theory of nonadiabatic electron transfer :(1985) Biochim. Biophys. Acta 811,2&L3221 leads to the conclusion that the SI! oxidation to S, is coupled with significant structural changes. Furthermore, the water oxidase in S3 is inferred to attain two different conformational states with populations that markedly change at a chrtracteristic transition temperature, PS II membrane fragment; Water oxidasc; Activation energy; Nonadiabatic electron transfer; S, conformation 1, INTRODUCTIONPhotosynthetic water oxidation to dioxygen and protons released into the thylakoid lumen takes place via a sequence of four univalent oxidation steps, comprising redox transitions at a manganese-containing unit (for recent reviews see [1,2]) Although the kinetics of the individual univalent redox steps have been unraveled by time resolved EPR-and W-spectroscopic measurements [3-51, the electronic configuration and the nuclear geometry of the intermediary redox states of the water oxidase are still unknown. To address this problem, two questions of central mechanistic relevance have to be answered: (i) are the univalent electron abstractions, symbolized by St + Sr+l, metal-or ligandcentered processes; and (ii) at which redox level does the key step of the overall reaction take place, i.e. the formation of an oxygen-oxygen bond? Based on our current knowledge it seems established that the S, + S2 redox transition is a manganese centered Mn(II1) + Mn(IV) oxidation whereas much less information is available on the other steps (for discussion see last univalent oxidation step although alternative mechanisms cannot be excluded [1,6]. Apart from spectral analyses, recent studies on the interaction of small redox active agents (NHzOH, NHzNH2) with the water oxidase led to the conclusion that the redox transition S1 + S3 is accompanied by a significant nuclear rearrangement [7,8].In this report results are presented on an attempt to gather further information on the reaction coordinates of the univalent redox steps in the water oxidase by determining the temperature dependence of their rate constants from measurements of 355 nm absorption changes induced by a train of laser flashes in dark adapted PS II membran fragments. The data obtained reveal: (i) the formation of S3 is coupled with significant structural changes; and (ii) the water oxidase in S3 und...

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The possibility to determine the difference spectra Δεi+1jλ of each univalent redox step Si→Si+1(i=0,...3) of the water-oxidizing enzyme system was analyzed by theoretical calculations and by measurements of 320 nm absorption changes induced by a train of saturating laser flashes (FWHM:7 ns) in PS II membrane fragments. It was found: a) Lipophilic quinones complicate the experimental determination of optical changes due the Si-state transitions because they lead to an additional binary oscillation probably caused by a reductant-induced oxidation of the Fe(2+) at the PS II acceptor side. b) In principle, a proper separation can be achieved at sufficiently high K3[Fe(CN)6] concentrations. c) An unequivocal deconvolution into the difference spectra Δεi+1jλ of flash train-induced optical changes which are exclusively due to Si-state transitions is impossible unless the Kok parameters α, β and [Si]0 can be determined by an independent method.Measurements of the oxygen yield induced by a flash train reveals, that in thylakoids and PS II membrane fragments Si is the stable state of dark adapted samples even at alkaline pH (up to pH=9). However, in PS II membrane fragments at pH>7.7 the misses probability α markedly increases, in contrast to the properties of intact thylakoids. Based on these data the possibility is discussed that an equilibrium exists of two types of S2-states with different properties.

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