Structural, Biochemical and Biophysical Characterization of Four Oxygen-Evolving Photosystem II Preparations From Spinach

Four procedures utilizing different detergent and salt conditions were used to isolate oxygen-evolving Photosystem II (PS II) preparations from spinach thylakoid membranes. These PS II preparations have been characterized by freeze-fracture electron microscopy, SDS-polyacrylamide gel electrophoresis, steady-state and pulsed oxygen evolution, 77 K fluorescence, and room-temperature electron paramagnetic resonance. All of the O₂-evolving PS II samples were found to be highly purified grana membrane fractions composed of paired, appressed membrane fragments. The lumenal surfaces of the membranes and thus the O₂-evolving enzyme complex, are directly exposed to the external environment. Biochemical and biophysical analyses indicated that all four preparations are enriched in the chlorophyll a b-light-harvesting complex and Photosystem II, and depleted to varying degrees in the stroma-associated components, Photosystem I and the CF₁-ATPase. The four PS II samples also varied in their cytochrome f content. All preparations showed enhanced stability of oxygen production and oxygen-rate electrode activity compared to control thylakoids, apparently promoted by low concentrations of residual detergent in the PS II preparations. A model is presented which summarizes the effects of the salt and detergent treatments on thylakoid structure and, consequently, on the configuration and composition of the oxygen-evolving PS II samples.