Elucidating the Water-Oxidation Mechanism in Natural Photosynthesis

Date of Award

Fall 10-1-2021

Document Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Brudvig, Gary


Natural photosynthesis is the only existing system of solar fuel production on a global scale; therefore, it provides a blueprint for developing environmentally friendly alternative energies. Photosynthesis is initiated by the water-oxidation reaction catalyzed by the protein complex photosystem II (PSII), in which the catalytic center is the oxygen-evolving complex (OEC). Understanding the water-oxidation reaction is crucial for making advances on global solar fuel production because this reaction produces abundant reducing equivalents for fuel production. However, the mechanism of the water-oxidation reaction in PSII is still unclear, partly due to the absence of the S4 state structure and the challenges in identifying the substrate waters. We used a variety of methods and techniques, from making steady-state oxygen assay and 18O kinetic isotope effect (18O KIE) measurements to analyzing kinetic models and cryo-electron microscopy (cryo-EM) data, in order to further our understanding on natural photosynthesis, with a focus on the mechanism of the water-oxidation reaction in PSII.Chapter 1 of this thesis is a comprehensive overview of the water-oxidation reaction and the current proposals of the reaction mechanism. We identified that the main missing piece is towards the end of the S-state cycle, namely the S3 to S0 transition, due to a lack of structural information of the S4 state. We decided to utilize 18O KIE measurements to probe this S3 to S0 transition without a need of trapping any intermediates. As the first step, we designed and optimized a vacuum manifold for effectively and reliably collecting dioxygen evolved from water-oxidation reactions. The home-built manifolds for 18O KIE measurements are presented in Chapter 2. With the optimized system in hand, we measured the 18O KIEs of PSII samples and some inorganic model complexes to probe their reaction mechanisms, as shown in Chapter 3. We obtained no 18O KIEs for the PSII samples, suggesting a rate-limiting step not involving substrate water. This conclusion will be an important experimental benchmark for computational studies proposing plausible pathways of the S3 to S0 transition. In Chapter 4, we used a different approach to understand the water-oxidation mechanism of PSII by studying the substrate waters. We constructed complex kinetic models to interpret recent substrate-water exchange kinetic data and surmised that the slowly-exchanging water (Ws) may be a water ligand of Mn4, namely W1 or W2. With the alternative interpretation, we pointed out that the original interpretation of assigning Ws to O5, albeit well-acknowledged, might still be debatable. We also used cryo-EM data to help us understand the water oxidation in PSII. In Chapter 5, we present a novel sodium-binding site near the OEC and a sodium dependence on oxygen-evolution activity. The discoveries provide a more complete picture of how the environment surrounding the OEC regulates the oxygen-evolution activity in PSII. Lastly, we shifted our focus from PSII to photosystem I acclimated to far-red light (FRL-PSI) and studied the chlorophyll (Chl) cofactors within it. As shown in Chapter 6, we developed a procedure to quantitatively assess the substituents on the Chl molecules to confidently distinguish Chl f from Chl a.

This document is currently not available here.