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Membranes and Bioenergetics

Characterization of the Activation of Membrane-Bound and Soluble CF₁ by Thioredoxin ¹

Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853

The activation of spinach (Spinacia oleracea) chloroplast coupling factor 1 (CF₁) by thioredoxin (ThR) was characterized using membrane-bound and soluble CF₁. Light generates an electrochemical proton gradient across the thylakoid membrane, which increases the accessibility of the disulfide bond on the -subunit of CF₁ to reduced ThR. The proton gradient substantially accelerates the activation of CF₁ compared with thylakoids incubated in the dark with similar concentrations of dithiothreitol and ThR. The interaction of soluble CF₁ with ThR was studied using fluorescent probes. CF₁ in solution, with and without its associated -subunit, was labeled at Cys-322 of the -subunit with fluoresceinyl maleimide. ThR from Escherichia coli was labeled with eosin isothiocyanate. Labeled ThR and CF₁ 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 CF₁ form an intermolecular complex. When the -subunit was absent, quenching of donor fluorescence was approximately doubled, indicating that labeled ThR could approach more closely to the -subunit of CF₁. The distance between the fluorescent probes on CF₁ and ThR was calculated to be approximately 65 Å when -subunit was present and 52 Å when was absent. These values are consistent with other distance measurements and energy transfer values reported previously for fluorescent probes on CF₁. Whereas the extent of quenching increased by removal of the -subunit, the apparent dissociation constant was unchanged. The quenching effect was reversed when the -subunit was added back to the titration mixture. Similarly, the addition of unlabeled ThR decreased donor quenching.

³ Present address: Department of Biology, The Johns Hopkins University, Baltimore, MD 21218.

¹ Supported in part by a grant from the National Science Foundation (DMB 88-03608) and by the Cornell University Biotechnology Program, which is sponsored by the New York State Science and Technology Foundation, a consortium of industries, the U.S. Army Research Office, and the National Science Foundation.

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