Activation energies for two steps in the S2 → S3 transition of photosynthetic water oxidation from time-resolved single-frequency infrared spectroscopy

The mechanism of water oxidation by the Photosystem II (PSII) protein-cofactor complex is of high interest, but specifically, the crucial coupling of protonation dynamics to electron transfer (ET) and dioxygen chemistry remains insufficiently understood. We drove spinach-PSII membranes by nanosecond-laser flashes synchronously through the water-oxidation cycle and traced the PSII processes by time-resolved single-frequency infrared (IR) spectroscopy in the spectral range of symmetric carboxylate vibrations of protein side chains. After the collection of IR-transients from 100 ns to 1 s, we analyzed the proton-removal step in the S-2 double right arrow S-3 transition, which precedes the ET that oxidizes the Mn4CaOx-cluster. Around 1400 cm(-1), pronounced changes in the IR-transients reflect this pre-ET process (similar to 40 mu s at 20 degrees C) and the ET step (similar to 300 mu s at 20 degrees C). For transients collected at various temperatures, unconstrained multi-exponential simulations did not provide a coherent set of time constants, but constraining the ET time constants to previously determined values solved the parameter correlation problem and resulted in an exceptionally high activation energy of 540 +/- 30 meV for the pre-ET step. We assign the pre-ET step to deprotonation of a group that is re-protonated by accepting a proton from the substrate-water, which binds concurrently with the ET step. The analyzed IR-transients disfavor carboxylic-acid deprotonation in the pre-ET step. Temperature-dependent amplitudes suggest thermal equilibria that determine how strongly the proton-removal step is reflected in the IR-transients. Unexpectedly, the proton-removal step is only weakly reflected in the 1400 cm(-1) transients of PSII core complexes of a thermophilic cyanobacterium (T. elongatus).