Water Oxidation Chemistry at the Oxygen-Evolving Complex in Photosystem II

The oxygen-evolving complex (OEC), a Mn4CaO 5 unit, in photosystem II (PSII) catalyzes light-driven water oxidation in oxygenic-photosynthetic organisms. The chloride ion is an essential cofactor for OEC turnover, but the role of chloride had not been previously determined. Depletion of chloride from PSII blocks some redox transitions of the OEC that are essential for water-oxidation catalysis. The blocked transitions are proton-coupled electron-transfer steps of the OEC. Chapter one of this thesis provides a general overview of oxygenic photosynthesis with a detailed review of the characterization of the OEC and important mechanistic insights regarding water oxidation in PSII. The biochemical and spectroscopic studies on the role of chloride are then reviewed in Chapter two. Based on the analysis of the mechanism by which chloride activates other chloride-dependent enzymes, a mechanism by which chloride activates PSII is proposed. The proposal unifies recent structural information on the binding site of chloride, gathered from recent higher-resolution crystal structures of PSII, with the available biochemical and spectroscopic data on chloride activation of PSII. In Chapter three, mutants from the cyanobacterium Synechocystis PCC 6803 are used to test the hypothesis presented in Chapter two. PSII isolated from wild type and mutants are characterized using steady-state oxygen-evolution assays, electron paramagnetic resonance spectroscopy (EPR), Fourier transform infrared (FTIR) spectroscopy, and xenon-flash induced oxygen polarographic measurements. The results in Chapter three support the proposal presented in Chapter two, and indicate that these mutations affect the chloride binding properties, the structural aspects of the OEC, the efficiency of redox transitions of the OEC, and the kinetics of oxygen release. We hypothesize that chloride is required for efficient proton transfer from the OEC to the lumen. Progress in the characterization of a different set of mutations close to the chloride site on the active face of the OEC is presented in Chapter four. In contrast to the mutants presented in Chapter three, the pathway of proton transfer from the OEC to the lumen is intact in these mutants, but catalysis is slowed which is proposed to be due to a unique impairment in the substrate water orientation. In Chapter five, investigation of the effect of nitrite on PSII is reported. Earlier work in the literature showed that nitrite can occupy the chloride-binding site in chloride-depleted PSII. A detailed study on the inhibitory effect of nitrite on PSII was carried out using PSII isolated from spinach. White we confirm that nitrite competes with chloride for the chloride-binding site, we also find that nitrite binds and reacts at additional sites in PSII, including the OEC. In Chapter six, we review the available EPR and FTIR data on the first oxidative transition of the OEC along with some high resolution crystal structures of the OEC. We propose that the hydrogen-bonding network plays an important role in stabilizing different spin isomers of the OEC. A mechanistic model for ammonia binding to the OEC is also presented. The findings in this thesis provide insight into the role of chloride in PSII and into the factors affecting turnover at the OEC.