Evidence for an association between a 33 kDa extrinsic membrane protein, manganese and photosynthetic oxygen evolution. I: Correlation with the S2 multiline EPR signal

Abstract The removal of peripheral membrane proteins of a molecular mass of 17 and 23 kDa by washing of spinach Photosystem-II (PS II) membranes in 1 M salt between pH 4.5 and 6.5 produces a minimal loss of the S 1 → S 2 reaction, as seen by the multiline EPR signal for the S 2 state of the water-oxidizing complex, while reversibly inhibiting O 2 evolution. The multiline EPR signal simplifies from a ‘19-line’ spectrum to a ‘16-line’ spectrum, suggestive of partial uncoupling of a cluster of 3 or 4 to yield photo-oxidation of a binuclear Mn site. Alkaline salt washing progressively releases a 33 kDa peripheral protein between pH 6.5 and 9.5, in direct parallel with the loss of O 2 evolution and the S 2 multiline EPR signal. The 33 kDa protein can be partially removed (20%) at pH 8.0 prior to managanese release. Salt treatment releases four Mn ions between pH 8.0 and 9.5 with the first 2 or 3 Mn ions released cooperatively. A common binding site is thus suggested in agreement with earlier EPR spectroscopic data establishing a tetranuclear Mn site. At least two of these Mn ions bind directly at a site in the PS II complex for which photooxidation by the reaction center is controlled by the 33 kDa protein. The washing of PS II membranes with 1 M CaCl 2 to affect the release of the 33 kDa protein, while preserving Mn binding to the membrane (Ono, T.-A. and Inoue, Y. (1983) FEBS Lett. 164, 255–260), is found to leave some 33 kDa protein undissociated in proportion to the extent of O 2 evolution and S 2 multiline yield. These depleted membranes do not oxidize water or produce the normal S 2 state without the binding of the 33 kDa protein. A method for the accurate determination of relative concentrations of the peripheral membrane proteins using gel electrophoresis is presented.