EXTENT AND RATE OF PROTON RELEASE BY PHOTOSYNTHETIC WATER OXIDATION IN THYLAKOIDS - ELECTROSTATIC RELAXATION VERSUS CHEMICAL PRODUCTION

The detailed chemical mechanism of the four steps of photosynthetic oxidation of two molecules of water to yield molecular oxygen plus four protons is under contention. The observed release of protons is a composite of the chemical production and more indirect reactions such as electrostatically induced shifts of acid/base equilibria of peripheral amino acids. In thylakoids we studied the extent and the rate (at microsecond time resolution) of proton release and uptake by each of the four oxidation steps. The pattern of net proton release in thylakoids varied drastically (between 0.3 and 2 H+/e-) as a function of pH. It differed substantially from the pH-dependent patterns of PSII-enriched membrane fragments and core particles, but the stepped progression toward release of dioxygen (the Kok parameter triple) was about the same. This implied an electrostatic origin of this variation and, with in the observed limits, a lack of (inhibitory) feedback of the uncompensated charge on the electrontransfer from the catalytic Mn cluster to Tyr(z)+. The rate of rapid proton transfer to the amphiphilic, surface-adsorbed indicator neutral red was proportional to its concentration. The shortest half-transfer time was 12 mus, substantially shorter than the time for electron transfer from Mn to Tyr(z)+ at any oxidation step. Rapid deprotonation thus occurred at the level of Tyr(z)+. By rapid deprotonation acts the four light-driven oxidation steps S0 double-line arrow pointing right S1 double-line arrow pointing right S2 double-line arrow pointing right S3 double-line arrow pointing right S4 created between 3.4 (at pH 7.4) and 4.5 (pH 6.3) bases per photosystem II. On the last step there was a compensating slow release of 0.6 proton (pH 7.4) and slow uptake of 0.5 proton (pH 6.3) in milliseconds, respectively. The slow event was attributed to the oxygen-evolving step, S4 --> S0. Our results are in favor of a concerted electron-proton transfer mechanism from water to a manganese-base cluster and are also in favor of one reaction step with water (S4 --> S0) as opposed to two two-electron reactions with a peroxide intermediate formed upon S1 double-line arrow pointing right S2.