5-Iodoacetamidofluorescein-labeled chloroplast coupling factor 1: conformational dynamics and labeling-site characterization

Physical and spectroscopic properties of 5-iodoacetamidofluorescein (5-IAF), a new sulfhydryl-specific fluorescent label, are described. Under certain conditions, 5-IAF labels the chloroplast phosphorylation coupling factor (CF) predominantly on the ..beta.. subunit. Approximately 88% of the ATPase activity and over 60% of the ability of CF to reconstitute photophosphorylation are retained after labeling. Trypsin activation of the ATPase activity of 5-IAF-labeled CF dramatically alters the fluorescence properties at the labeling site, indicating its involvement in ATPase activation. Studies of the fluorescence emission spectra, fluorescence polarization, and potassium iodide quenching of 5-IAF and 5-IAF-labeled CF demonstrate that the labeling site is in a partially buried hydrophobic region which is partially accessible to potassium iodide quenching from the solvent phase and which restricts the motion of the fluorescent label. The fluorescence shows little change upon substrate binding. We conclude that the label is located in a cleft region remote from the enzyme active site. Variation of the reaction pH between 6.4 and 8.5 significantly alters the number of attached labels. ATP decreases the extent of labeling over the entire pH range studied. These labeling changes reflect substrate- and pH-induced conformational changes in CF. Certain interdependences observed in these conformational changes suggest that the transmembrane electrochemical gradient may directly induce conformational changes in CF leading to net ATP synthesis. The ..gamma.. subunit plays a central role in the expression of these intersubunit conformational changes.