A sensitive and simple reverse phase HPLC purification scheme was developed for the rapid separation of the small protein subunits from photosystem II reaction center preparations. The precise molecular masses of the ␣-and -subunits of cytochrome b 559 and the psbI gene product from pea plants, found to be 4394.6 ؎ 0.6, 9283.6 ؎ 0.7, and 4209.5 ؎ 0.5 Da, respectively, were then successfully determined for the first time by electrospray-and fast atom bombardment-mass spectrometry. Discrepancies between the molecular weights assigned and those calculated from the respective DNA sequences were observed for ␣-and -subunits of cytochrome b 559 . Currently, the nucleotide sequence of the psbI gene product from pea plants is not available. Application of novel mapping and sequencing strategies has assured the elucidation of full primary structures of all of the purified subunits. The modifications identified here include the post-translational processing of the initiating methionine on both subunits of cytochrome b 559 , NH 2 -terminal acetylation and an mRNA editing site at residue 26 (Ser 3 Phe) on the -subunit, and retention of the NH 2 -terminal formyl-Met on the psbI gene product. In addition, specific oxidation of a single amino acid residue was identified on the psbI gene product and the -subunit purified from light-treated reaction center preparations. Overall, these studies provide the first detailed primary structural characterization of the small subunits of the reaction center complex and their associated light-induced modifications.
Photosystem II (PSII)1 catalyzes the photoinduced splitting of water into molecular oxygen and reducing equivalents. This complex is embedded in the thylakoid membrane of plants, algae, and cyanobacteria and is composed of a large number of subunits (1, 2). At the heart of the complex is the reaction center where primary and secondary electron transfer processes occur. The cofactors that support these reactions are bound to the D1 and D2 proteins. These proteins are the products of the psbA and psbD genes, respectively, and are comparable with the L and M subunits of the reaction center of purple photosynthetic bacteria (3).In its isolated form, the reaction center of PSII contains, in addition to the D1 and D2 proteins, the ␣-and -subunits of cytochrome b 559 , which are the products of the psbE and psbF genes, and the PSII-I protein, which is encoded by the psbI gene. In a recent paper (4), a sixth component has been claimed, which has an apparent molecular mass of about 6.5 kDa and is the product of the nuclear gene recently named psbW.Sites in the D2 and D1 proteins, named Q A and Q B respectively, bind plastoquinones that facilitate secondary electron flow. However, in the case of the isolated reaction center complex the plastoquinone binding affinity is significantly reduced, and no secondary electron flow occurs unless appropriate acceptors are added (5). In their absence, the photochemical reaction is restricted to the formation of the radical pair state P680 ϩ Phe...