CHEMICAL-REDUCTION OF THE WATER-SPLITTING ENZYME-SYSTEM OF PHOTOSYNTHESIS AND ITS LIGHT-INDUCED REOXIDATION CHARACTERIZED BY OPTICAL AND MASS-SPECTROMETRIC MEASUREMENTS - A BASIS FOR THE ESTIMATION OF THE STATES OF THE REDOX-ACTIVE MANGANESE AND OF WATER IN THE QUATERNARY OXYGEN-EVOLVING S-STATE CYCLE

The chemical reduction and over-reduction of the water-splitting enzyme and its subsequent light-induced reoxidation was followed by simultaneous measurements of (i) UV and (ii) electrochromic absorption changes, accompanied (iii) by O2 measurement and (iv) mass spectrometric analysis of the evolved N-15(2) and (N2O)-N-15 of the applied reductant, (NH2OH)-N-15. (1) On this reversible pathway, the measured four characteristics attributed to changes of Mn valences, surplus charges, water oxidation and oxidation of the reductant are quantitatively coupled with each other. (2) Treatment with NH2OH (or NH2NH2) leads to a three-digit reverse shift of O2 evolution and UV and electrochromic absorption changes. This is interpreted as the reduction of the enzyme in the dark from the S1 state via the S0 and the overreduced S-1 state to the superreduced state, S-2. (3) S-2 is stable for hours. (4) S-2 is a terminal state, not further reducible without being deactivated. (5) Each step on this reversible pathway from state S1 to S-2 is accompanied by a 1/2-N-15(2) evolution from (NH2OH)-N-15 interpreted as a three-electron reduction of state S1 which is in accordance with the three-digit reverse shifts. (6) The results are explained by a reversible reduction of 3 redox-active Mn ions. Based on the obtained data, the possible valence states of these photooxidizable Mn ions in state S-2 to S3 have been estimated. (7) Irreversible deactivation of S-2 with release of Mn into the solution is coupled with an approx. 1-(N2O)-N-15 evolution from (NH2OH)-N-15. (8) The electrochromic changes indicate at pH 7 a surplus charge at S-1, S2 and S3. This is explained by an H+ release of approx. 2:1:0:1 with the 1:1:1:1 electron extraction in the S-1 --> S0 --> S1 --> S2 --> S3 transition. On this basis, the possible states of the water derivatives up to S3 have been estimated. (9) The results are compared with information obtained from UV-difference spectra and XAS, EPR and NMR measurements.