Thiol modulation of the thylakoid ATPase. Lack of oxidation of the enzyme in the presence of [formula omitted] in vivo and a possible explanation of the physiological requirement for thiol regulation of the enzyme

Abstract

(1) Illumination of Dunaliella induced an increase in the activity of CF 1-ATPase, and of fructose-1,6-bisphosphatase (FBPase), that could be assayed under standard conditions in subsequently lysed cells. The light-induced, but not the light-independent, activities could be prevented by inclusion of methyl viologen in the preillumination medium. This effect was concluded to be due to the prevention of reduction of ferredoxin by methyl viologen. (2) Markedly higher concentrations of added methyl viologen were required to prevent induction of ATPase than FBPase. Addition of 1 mM methyl viologen to the preillumination stage prior to illumination totally prevented the appearance of light-induced FBPase, whereas a concentration of 20 mM was required to prevent light-induction of ATPase completely. (3) Although methyl viologen added to intact algae in the light subsequent to achievement of steady-state light activation of ATPase and FBPase led to the inactivation of FBPase, light-induced ATPase was completely uninhibited. This effect indicates that whereas FBPase is subject to reversible modification by thiols in the light, thiol reduction of ATPase in vivo is irreversible in the light, and oxidation only occurs upon dissipation of Δ μ H + produced by darkening. (4) Stabilisation of the thiol-reduced form of CF 1 by Δ μ H + (Shahak, Y. (1985) J. Biol. Chem. 260, 1459–1464) accounts for the lower reducing pressure necessary to activate thiol-dependent ATPase, compared to FBPase. Implications for the regulation of CO 2 fixation are discussed. (5) Complete activation of CF 1-ATPase activity was induced by incubation of intact algae with dithiothreitol in the dark. It is suggested that thiol-regulation of the enzyme is necessary in vivo in order to prevent activation of an ATPase activity which would otherwise inevitably result from autocatalytic generation of Δ μ H + in the dark.