Spectroscopic investigations of photosynthetic water oxidation in photosystem II

Photosystem II (PSII) catalyzes photosynthetic water oxidation in plants, green algae, and cyanobacteria. The manganese-containing active site cycles through a series of five oxidation states, S_n, where n refers to the number of oxidizing equivalents stored. This thesis describes the effects of dark adaptation and addition of sucrose and glycerol on photosynthetic water oxidation. Structural differences in each of the S-states are also identified through the use of Fourier transform infrared (FT-IR) spectroscopy. This thesis research produced three unexpected results. First, dark adaptation effects observed for the S₁ to S₂ transition suggest changes in carboxylate ligation to manganese and/or changes in secondary structure occur during dark adaptation in the S₁ state. This is the first description of structural changes defining a new S₁ ′ intermediate, at the same oxidation level as S₁. The second novel result is the different effects of sucrose and glycerol on PSII. The addition of sucrose stimulated the steady state rate of oxygen evolution in both intact PSII and PSII samples treated to remove the extrinsic subunits. However, the addition of glycerol stimulated oxygen evolution for intact PSII only. The data showed that both sucrose and glycerol were inhibitory in all samples at concentrations higher than 1.35 M or 30% respectively. The data suggests that the observed activity stimulation is a result of structural changes in the active site. These structural changes can be attributed to a change in preferential hydration of PSII. Glycerol inhibition is attributed to glycerol binding to the active site when the extrinsic subunits are not present. A unique procedure for acquiring time-resolved reaction-induced difference FT-IR spectra for each of the S-state transitions was also developed. Double difference spectra constructed for the S₃/S₂ and the S₄-S₀/S₃ transitions suggest that protein conformation is relatively insensitive to Mn-oxidation up to the generation of the S₃ state. However, more substantial structural changes occur during the S₃ to S₄-S₀ transition. The data presented in this thesis demonstrates the importance of protein flexibility and protein structure on the efficient catalysis of water oxidation by Photosystem II.