Final Activity Report Summary - O2FROMPSII (How plants can live on solar energy and water. Biophysical investigations of O2 evolution in Photosystem II, the key reaction in photosynthesis)
Photosynthesis is the key to life on earth as we know it. Not only is it the energy source of plants, algae and certain bacteria, which form the ultimate basis of the food chain in the complex ecosystems of our planet, oxygenic photosynthesis from cyanobacteria and plants also produce the oxygen that we breathe. This is achieved by the splitting of water into oxygen and protons, an energetically extremely demanding process that these organisms are remarkably able to perform at mild physiological conditions.
This is made possible by a protein called Photosystem II (PSII), and this is where the engine for water splitting, the oxygen evolving centre (OEC), is found. It consists of a metal cluster, CaMn4, and a nearby redox active tyrosine, YZ. It is the CaMn4 cluster that ultimately extracts electrons from water during water splitting. Despite many years of research, neither the mechanism by which this occurs, nor the structure of the cluster, are precisely known. Our research has focussed on studying the flow of proton and electrons within the OEC better understand this most important of reactions.
During water splitting the CaMn4 is oxidised stepwise in the so-called 'S-cycle'. We have developed a technique using Electron paramagnetic resonance (EPR) spectroscopy at room temperature to monitor the changes in the cluster in real-time. Traditional techniques have relied on cryogenic temperatures to slow down or freeze in extremely rapid reactions that take place, but we have been able to exploit another important tyrosine in PSII, YD, to follow the formation and decay of the various steps of the S-cycle (the 'S-states'). We found that the relaxation properties of YD also changed in an S-state dependent manner, which allowed YD to used as a new probe for studying the mechanistic details of the S-cycle. Being at room temperature, this technique gives a dynamic picture of the reactions under physiological conditions. It is a new tool in the arsenal of spectroscopic techniques for discovering the structural and mechanistic details of the OEC.
Cryogenic studies, however, are also important, trapping unstable states and species. We have extensively studied the so-called EPR 'split signals', believed to arise from the interaction of the CaMn4 cluster with the YZ* radical, generated and trapped by illumination at < 20 K. These signals are distinct for each S-state, reflecting changes in the magnetic properties of the cluster. We have demonstrated that these signals respond to changes in the environment, revealing important information about how electron and proton transfers take place, and how substrate molecules interact with the cluster. For instance, methanol, a molecule closely related to water, causes significant changes in these split signals. By combining experimental concentration dependence data with a theoretical framework for the signals' origins, we were able to deduce that the magnetic coupling between the Mn ions were changed by the binding of methanol to the same Mn ion of the cluster in all S-states for which split signals were known. Molecular modelling based on the best PSII structure currently available supported this conclusion, and identified which of the Mn ions is likely to be the binding site. This investigation revealed moreover channels within PSII which may act as conduits for the approach of methanol and water to the CaMn4 cluster, and critical regions in these channels were bordered by amino acid residues known to be of mechanistic importance.
Complementary studies using advanced optical spectroscopic studies have also been performed in collaboration with a leading group in Australia. We have obtained kinetic data of various redox active species under split signal induction conditions, as well as PSII spectra under various conditions, including the first known S-state dependent optical spectra. We have also seen indications of S-state dependent shifts in spectral bands as a result of split signal inducing illuminations. Combined with EPR data, we are ever closer to discovering the minutiae of how plants can live on sunlight and water.
This is made possible by a protein called Photosystem II (PSII), and this is where the engine for water splitting, the oxygen evolving centre (OEC), is found. It consists of a metal cluster, CaMn4, and a nearby redox active tyrosine, YZ. It is the CaMn4 cluster that ultimately extracts electrons from water during water splitting. Despite many years of research, neither the mechanism by which this occurs, nor the structure of the cluster, are precisely known. Our research has focussed on studying the flow of proton and electrons within the OEC better understand this most important of reactions.
During water splitting the CaMn4 is oxidised stepwise in the so-called 'S-cycle'. We have developed a technique using Electron paramagnetic resonance (EPR) spectroscopy at room temperature to monitor the changes in the cluster in real-time. Traditional techniques have relied on cryogenic temperatures to slow down or freeze in extremely rapid reactions that take place, but we have been able to exploit another important tyrosine in PSII, YD, to follow the formation and decay of the various steps of the S-cycle (the 'S-states'). We found that the relaxation properties of YD also changed in an S-state dependent manner, which allowed YD to used as a new probe for studying the mechanistic details of the S-cycle. Being at room temperature, this technique gives a dynamic picture of the reactions under physiological conditions. It is a new tool in the arsenal of spectroscopic techniques for discovering the structural and mechanistic details of the OEC.
Cryogenic studies, however, are also important, trapping unstable states and species. We have extensively studied the so-called EPR 'split signals', believed to arise from the interaction of the CaMn4 cluster with the YZ* radical, generated and trapped by illumination at < 20 K. These signals are distinct for each S-state, reflecting changes in the magnetic properties of the cluster. We have demonstrated that these signals respond to changes in the environment, revealing important information about how electron and proton transfers take place, and how substrate molecules interact with the cluster. For instance, methanol, a molecule closely related to water, causes significant changes in these split signals. By combining experimental concentration dependence data with a theoretical framework for the signals' origins, we were able to deduce that the magnetic coupling between the Mn ions were changed by the binding of methanol to the same Mn ion of the cluster in all S-states for which split signals were known. Molecular modelling based on the best PSII structure currently available supported this conclusion, and identified which of the Mn ions is likely to be the binding site. This investigation revealed moreover channels within PSII which may act as conduits for the approach of methanol and water to the CaMn4 cluster, and critical regions in these channels were bordered by amino acid residues known to be of mechanistic importance.
Complementary studies using advanced optical spectroscopic studies have also been performed in collaboration with a leading group in Australia. We have obtained kinetic data of various redox active species under split signal induction conditions, as well as PSII spectra under various conditions, including the first known S-state dependent optical spectra. We have also seen indications of S-state dependent shifts in spectral bands as a result of split signal inducing illuminations. Combined with EPR data, we are ever closer to discovering the minutiae of how plants can live on sunlight and water.