Proton gradient across the chloroplast thylakoid membrane governs the redox regulatory function of ATP synthase

Chloroplast ATP synthase (CF_oCF₁) synthesizes ATP by using a proton electrochemical gradient across the thylakoid membrane, termed ΔμH⁺, as an energy source. This gradient is necessary not only for ATP synthesis but also for reductive activation of CF_oCF₁ by thioredoxin, using reducing equivalents produced by the photosynthetic electron transport chain. ΔμH⁺ comprises two thermodynamic components: pH differences across the membrane (ΔpH) and the transmembrane electrical potential (ΔΨ). In chloroplasts, the ratio of these two components in ΔμH⁺ is crucial for efficient solar energy utilization. However, the specific contribution of each component to the reductive activation of CF_oCF₁ remains unclear. In this study, an in vitro assay system for evaluating thioredoxin-mediated CF_oCF₁ reduction is established, allowing manipulation of ΔμH⁺ components in isolated thylakoid membranes using specific chemicals. Our biochemical analyses revealed that ΔpH formation is essential for thioredoxin-mediated CF_oCF₁ reduction on the thylakoid membrane, whereas ΔΨ formation is nonessential.