Characterization of the low molecular weight photosystem II reaction center subunits and their light-induced modifications by mass spectrometry.

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 alpha- and beta-subunits of cytochrome b559 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 alpha- and beta-subunits of cytochrome b559. 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 b559, NH2-terminal acetylation and an mRNA editing site at residue 26 (Ser --> Phe) on the beta-subunit, and retention of the NH2-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 beta-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.