Cytochrome b559 is an essential component of the photosystem II (PSII) protein complex. Its function, which has long been an unsolved puzzle, is likely to be related to the unique ability of PSII to oxidize water. We have used EPR spectroscopy and spectrophotometry redox titrations to probe the structure of cytochrome b559 in PSII samples that have been treated to remove specific components of the complex. The results of these experiments indicate that the low-temperature photooxidation of cytochrome b559 does not require the presence of the 17-, 23-, or 33-kDa extrinsic polypeptides or the Mn complex (the active site in water oxidation). We observe a shift in the g value of the EPR signal of cytochrome b559 upon warming a low-temperature photooxidized sample, which presumably reflects a change in conformation to accommodate the oxidized state. At least three redox forms of cytochrome b559 are observed. Untreated PSII membranes contain one high-potential (375 mV) and one intermediate-potential (230 mV) cytochrome b559 per PSII. Thylakoid membranes also appear to contain one high-potential and one intermediate-potential cytochrome bi59 per PSII, although this measurement is more difficult due to interference from other cytochromes. Removal of the 17- and 23-kDa extrinsic polypeptides from PSII membranes shifts the composition to one intermediate-potential (170 mV) and one low-potential (5 mV) cytochrome b559. This large decrease in potential is accompanied by a very small g-value change (0.04 at gz), indicating that it is the environment and not the ligand field of the heme which changes significantly upon the removal of the 17- and 23-kDa polypeptides. Removal of the 33-kDa polypeptide has no effect on the reduction potential of cytochrome b559; removal of the Mn complex may cause a small decrease in the potential. We propose that the 17-and 23-kDa polypeptides stabilize a very hydrophobic heme environment, which is responsible for the unusually high potential of the high-potential form of cytochrome b559. The lower potential forms of cytochrome b559 that result when the extrinsic polypeptides are removed probably reflect a conformation in which the heme is more accessible to the solvent. Our results demonstrate the importance of performing redox titrations to identify the forms of cytochrome b559 that are present in a PSII sample and indicate that the intermediate-and low-potential forms are not heterogeneous, denatured forms of cytochrome b559.
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