Characterization of the Activation of Membrane-Bound and Soluble CF(1) by Thioredoxin.
The activation of spinach (Spinacia oleracea) chloroplast coupling factor 1 (CF(1)) by thioredoxin (ThR) was characterized using membrane-bound and soluble CF(1). Light generates an electrochemical proton gradient across the thylakoid membrane, which increases the accessibility of the disulfide bond on the gamma-subunit of CF(1) to reduced ThR. The proton gradient substantially accelerates the activation of CF(1) compared with thylakoids incubated in the dark with similar concentrations of dithiothreitol and ThR. The interaction of soluble CF(1) with ThR was studied using fluorescent probes. CF(1) in solution, with and without its associated epsilon-subunit, was labeled at Cys-322 of the gamma-subunit with fluoresceinyl maleimide. ThR from Escherichia coli was labeled with eosin isothiocyanate. Labeled ThR and CF(1) showed normal activities. Fluorescence energy transfer between donor fluoresceinyl maleimide and acceptor eosin isothiocyanate, manifested by a quenching of the donor fluorescence, was detected, suggesting that ThR and CF(1) form an intermolecular complex. When the epsilon-subunit was absent, quenching of donor fluorescence was approximately doubled, indicating that labeled ThR could approach more closely to the gamma-subunit of CF(1). The distance between the fluorescent probes on CF(1) and ThR was calculated to be approximately 65 A when epsilon-subunit was present and 52 A when epsilon was absent. These values are consistent with other distance measurements and energy transfer values reported previously for fluorescent probes on CF(1). Whereas the extent of quenching increased by removal of the epsilon-subunit, the apparent dissociation constant was unchanged. The quenching effect was reversed when the epsilon-subunit was added back to the titration mixture. Similarly, the addition of unlabeled ThR decreased donor quenching.
Dann
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Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
McCarty
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RE