ANALYTICAL PROCEDURES FOR THE QUANTIFICATION OF ISOTOPIC AMINO-ACID-INCORPORATION INTO PHOTOSYNTHETIC PROTEINS OF SYNECHOCYSTIS PCC-6803

The mechanism of oxygen evolution has been an enigma for nearly two ce nturies. Pioneering work by Bessel Kok, Pierre Joliot, and many others during the last quarter century has provided valuable insight into th is most unique and important chemical reaction. The late 1970s and ear ly 1980s saw the introduction of biochemical techniques for the purifi cation of photosynthetic complexes that have, in turn, stimulated the biophysical chemists and spectroscopists to apply high resolution tech niques in order to resolve the structure/function relationships in the se protein complexes. Valuable information about events at the atomic level can be gained through isotopic substitution of particular amino acids thought to be important in the catalytic process. The ability to generate functional auxotrophs in the photosynthetic cyanobacterium S ynechocystis 6803 has been used successfully to identify the redox act ive components Z and D as tyrosine residues in the reaction center of Photosystem II. In this report, we present results of the application of specific isotopic labeling for high resolution spectroscopy of puri fied PS II particles. We have developed analytical procedures for moni toring the incorporation of both H-2 and O-17 labeled amino acids by g as chromatography-mass spectroscopic analysis. We also show that the g rowth curve of cells subjected to obligate auxotrophy displays two dis tinct stationary phases; one that corresponds to depletion of exogenou s amino acids, and a second that corresponds to the normal cell densit y at stationary phase. Cells harvested at the second stationary phase show little or no retention of the labeled amino acid.