The Oxygen Evolving cycle in Photosystem II studied with EPR Spectroscopy at different pH and Temperature

PSII catalyses the light-induced water splitting process which leads to electron transfer through the thylakoid membrane and formation of energy rich molecules. Several redox components including a Mn4Ca, an amino acid residueYZ, two special Chl molecules P680, two Pheo and two quinone molecules participate in this process. Another component of PSII which interacts magnetically with Mn4Ca is D2-Tyr161 and is positioned symmetrically with YZ to P680+. The active site of this enzyme is the Mn4Ca cluster where two substrate water molecules bind/ oxidize, and an O2 is released. The Mn4Ca cluster oscillates through 5 semi-stable intermediate oxidation states (Si, i = 0-4), where the S1 state is the most dark-stable state and the S4 state is a transient state, where O2 is released.

The EPR, laser flash and low temperature freezing techniques have been used to study the properties of semi-stable states of the Mn4Ca cluster at different pH and temperatures under experimental conditions.

The flash-induced Si state turnover at different pHs revealed transition inhibition at extreme pH. The inhibition pKs in the acidic region are 4.0, 4.5 and 4.7 for S2/S3, S3/S0 and S0/S1 respectively. The inhibition pKs at alkaline pH are 9.4 and 8.0 for S2/S3 and S3/S0 respectively. The S0/S1 and S1/S2 transition are open at alkaline and both acidic and alkaline pH respectively. We suggest the inhibition pKs at acidic pH are due to: (1) protonation of carboxylic residues ligand to the Mn4Ca cluster and function at the proton transfer pathway from active site to the lumen; (2) increasing of YZ/YZox redox potential close to P680 by lowering pH where P680+ is no longer able to oxidize Yz.

The inhibition of Si state turnover at alkaline pH is suggested to be due to reduction of the YZ/Yzox couple redox potential close to S3/S2 thereby unable to oxidize S2 state. This shift the equilibrium S3YZ ? S2YZ? to the right, radical split signal at high pH induced. The signal intensity increased with increasing pH to a maximum value around pH 8.5-8.9. A single pK was determined to be around 8.5. We suggest this proton originates from the His-YZ moiety and the YZ radical is trapped at high pH due to proton deficiency and lowering of Yz/YZox reduction potential.

The oscillation of Si state of the Mn4Ca cluster at different temperatures at pH 6 revealed two types of miss: (1) real misses which have mechanistic origin are where no charge separation occurs upon the applied flash of light; (2) apparent misses, where a flash-induced charge-separated state of the reaction centre is lost due to recombination reactions at higher temperature. At optimal temperature, where the apparent misses are close to zero the misses are very Si state-dependent and they only originate from higher Si states. This is consistent with the higher activation energy of the Si state turnover at the higher oxidation state of the Mn4Ca.

The YD residue is known to interact magnetically with the Mn4Ca at cryogenic temperature. We utilized the relaxation property of the YD radical at the S1 and S2 state to study S2 state decay kinetics after flash-induced S2 state formation at room temperature. The decay kinetics were found to be biphasic in the presence of both PpBQ and DCMU. The half- life time with PpBQ was about 0.7 s (10-15% of total amplitude) and 4 minutes (85-90%) and in the presence of DCMU was about 0.23 s (about 30%) and 1.8 s (70%).