PARENT SESSION

Symposium S7A Mechanisms of water oxidation
Thursday September 2nd, 2004 2:40 PM-4:40 PM Room 210A
Chair: Stenbjörn Styring
Co-Chair: Ron Pace

Photosynthetic water oxidation driven backward. Jürgen Clausen¹, Wolfgang Junge^*,2, ¹ Biophysik, Universität Osnabrück, Osnabrück, Germany² Biophysik, Universität Osnabrück, Osnabrück, Germany

ABSTRACT- Powered and clocked by quanta of light the catalytic centre of water oxidation accumulates four oxidising equivalents before oxygen is released. The first three equivalents are stored on the Mn₄Ca-cluster, raising its formal oxidation state from S₀ to S₃, and the third on Y_Z, producing S₃Y_Z^ox. Only thereafter water oxidation,implying transfer of four electrons, proceeds in what appears as a single reaction step (S₃Y_Z^ox(H₂O)₂+ O₂+4H⁺+S₀). Intermediate oxidation products of bound water have not yet been detected. We followed two experimental strategies to track down intermediate reaction products (e.g. bound peroxide): (i) kinetically, by high time resolution of oxygen evolution, and (ii) thermodynamically, by enhanced oxygen pressure. (i) PS-core particles were centrifuged on a kinetically competent Pt-electrode (rise time 100 s). The half-rise times of oxygen release were 1.35 ms (20°C, Synechocystis WT*) and 13.1 ms (point mutant D1-D61N), yielding a perfectly linear Arrhenius plot between -2° and 32°C. A short lag phase of the polarographic transient (duration at 20°C: 0.45 ms, activation energy: 31 kJ/mol) was absent in the UV-transients attributable to the transition S₃ → S₀ of Mn₄Ca (at 360 nm). Thus this intermediate, probably transiently bound oxygen, was not interesting. (ii) We attempted to block oxygen evolution by a shift of the equilibrium between any putative intermediate and the product state (O₂+4H⁺+S₀Y_Z) by increasing the oxygen pressure from 0.21 (i.e. ambient) up to 30 bar. The reduction of the Mn₄Ca-cluster after the third flash of light given to dark adapted PSII-cores was photometrically monitored at a wavelength of 360 nm. The extent of the negative absorption transient, which is generally attributed to the reduction of S₃ to S₀, decreased with increasing oxygen pressure. Half-suppression occurred at the rather low oxygen pressure of 2.3 bar. Hydrostatic pressure alone (19 bar N2) was without any effect. We interpreted these data in terms of a stabilized peroxide intermediate and calculated the standard free-energy profile of the tentative reaction sequence S₃Y_Z^ox(H₂O)₂ → S₂Y_Z(H₂O₂) (+1.7 kJ/mol), and S₂Y_Z(H₂O₂) → S₀Y_Z + O₂ + protons (-4.7 kJ/mol). This is the first experimental evidence for an intermediate oxidation product of bound water.

KEY WORDS: oxygen evolution, peroxide, water oxidation, manganese