Spin-probes designed for measuring the intrathylakoid pH in chloroplasts.

Abstract Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with p K values in the range 4.7–7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1 H -imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1 H -imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1 H -imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pH in established during illumination of chloroplasts with continuous light. For isolated bean ( Vicia faba ) chloroplasts suspended in a medium with pH out  = 7.8, we found that pH in  ≈ 5.4–5.7 in the state of photosynthetic control, and pH in  ≈ 5.7–6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference ΔpH ≈ 1.8–2.1. These values of ΔpH are consistent with a point of view that under steady-state conditions the proton gradient ΔpH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H + /ATP is n  ≥ 4–4.7.