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 centuries. Pioneering work by Bessel Kok, Pierre Joliot, and many others during the last quarter century has provided valuable insight into this most unique and important chemical reaction. The late 1970s and early 1980s saw the introduction of biochemical techniques for the purification of photosynthetic complexes that have, in turn, stimulated the biophysical chemists and spectroscopists to apply high resolution techniques in order to resolve the structure/function relationships in these 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 Synechocystis 6803 has been used successfully to identify the redox active 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 purified PS II particles. We have developed analytical procedures for monitoring the incorporation of both ²H and ¹⁷O labeled amino acids by gas chromatography-mass spectroscopic analysis. We also show that the growth curve of cells subjected to obligate auxotrophy displays two distinct stationary phases; one that corresponds to depletion of exogenous amino acids, and a second that corresponds to the normal cell density at stationary phase. Cells harvested at the second stationary phase show little or no retention of the labeled amino acid.Abbreviations D1 D2 reaction center core proteins of Photosystem II encoded by the psbA and psbD genes, respectively - FTIR Fourier transform infrared - ENDOR electron-nuclear double resonance - ESEEM electron spin-echo envelope modulation - EXAFS extended X-ray absorption fine structure - iBuCF isobutylchloroformate - OD optical density - XANES X-ray absorption near-edge structure - Y_D Y_Z redox active tyrosines of PS II