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

Abstract

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.

Molecular Dissection of the [epsilon] Subunit of the Chloroplast ATP Synthase of Spinach

“…The changes observed with the y subunit include heightened sensitivity of y to trypsinization (Moroney and McCarty, 1982;Schumann et al, 1985) and greater accessibility of Cys8' to chemical modification (McCarty and Fagan, 1973). In addition, the accessibility of the y disulfide to reducing agents, such as DTT and thioredoxin, increases (Dann and McCarty, 1992). A similar increase is also observed when E is extracted from CF,CFo.…”

Mechanism of Activation of the Chloroplast ATP Synthase

“…It was also reported that the presence of the ⑀ subunit increases the distance between ␥-Cys 322 and Trx in the ATPase⅐Trx supercomplex without changing its dissociation constant (23). In the present work, the kinetic study of the reduction of intact CF 1 by Trx was complicated by the release of the ⑀ subunit.…”

“…Surprisingly, it reports for the first time kinetic data about the interaction between Trx and the isolated CF 1 (with or without its ⑀ subunit). Previous data included qualitative studies of the interaction between CF 1 and different Trxs (22,23), quantitative studies of this interaction at equilibrium using fluorescent probes (23), and kinetic studies of this interaction in thylakoids (21). These different approaches are complementary.…”

“…However, Trx m is also able to reduce CF 1 with a good efficiency (21). Escherichia coli Trx, which is more similar in primary structure to Trx m than to Trx f, is more efficient than the former toward CF 1 reduction and has already been used for in vitro studies of its interaction with CF 1 (21)(22)(23). Binding properties of E. coli Trx to the isolated CF 1 subcomplex, devoid or not of its ⑀ inhibitory subunit, and in different redox states, have been investigated using fluorescent techniques (23).…”

“…Escherichia coli Trx, which is more similar in primary structure to Trx m than to Trx f, is more efficient than the former toward CF 1 reduction and has already been used for in vitro studies of its interaction with CF 1 (21)(22)(23). Binding properties of E. coli Trx to the isolated CF 1 subcomplex, devoid or not of its ⑀ inhibitory subunit, and in different redox states, have been investigated using fluorescent techniques (23). The kinetics of thiol modulation of CF 0 CF 1 by different Trxs in energized thylakoid membranes has been analyzed and the rate constant of binding determined in the presence of a high protonmotive force (21).…”