Selective modification of coupling factor 1 in spinach chloroplast thylakoids by a fluorescent maleimide.
N-(1-Anilinonaphthyl-4)maleimide (ANM) has been used to modify coupling factor 1 (CF1), the terminal coupling factor of photophosphorylation in chloroplasts. As with other monofunctional maleimides, incubation of thylakoids with ANM in the light, but not in the dark, causes energy transfer inhibition of photophosphorylation. In the dark, sites on both the gamma and epsilon subunits of CF1 are modified. The light-accessible site is also on the gamma subunit. Trypsin digestion of the enzyme after dithiothreitol activation reveals that the dark-and light-accessible sites on the gamma subunit are different amino acid residues. Fluorescence of ANM bound at the dark-and light-accessible sites has been measured after isolation of CF1 from thylakoids. The fluorescence emission maximum of ANM at the light-accessible site is blue-shifted and the quantum yield is increased 2-fold relative to ANM bound at dark-accessible sites. On the soluble enzyme, fluorescence polarization is high and equivalent for ANM bound at both dark-and light-accessible sites. Fluorescence energy transfer from a tryptophan in a hydrophilic region of the epsilon subunit to ANM bound to the epsilon subunit but not to the gamma subunit has been observed. The significance of these observations is discussed with respect to the structure of the gamma subunit and its role in conformational transitions within CF1 that occur during energization of the membrane.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002736:Chloroplasts / Q000187:drug effects / Q000378:metabolism; D007202:Indicators and Reagents / Q000494:pharmacology*; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology*; D010785:Photophosphorylation / Q000187:drug effects*; D010944:Plants / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D007202:Indicators and Reagents; D008301:Maleimides; C010854:N-(1-anilinonaphthalyl)-4-maleimide; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D006180:Proton-Translocating ATPases; D000252:Calcium-Transporting ATPases