Spin-probes designed for measuring the intrathylakoid pH in chloroplasts

Alexander N. Tikhonov, Roman V. Agafonov, Igor A. Grigor'ev, Igor A. Kirilyuk, Vasilii V. Ptushenko, Boris V. Trubitsin

Research output: Contribution to journalArticlepeer-review

40 Citations (Scopus)


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 pK 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-1H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1H-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 pHin established during illumination of chloroplasts with continuous light. For isolated bean (Vicia faba) chloroplasts suspended in a medium with pHout = 7.8, we found that pHin ≈ 5.4-5.7 in the state of photosynthetic control, and pHin ≈ 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.

Original languageEnglish
Pages (from-to)285-294
Number of pages10
JournalBiochimica et Biophysica Acta - Bioenergetics
Issue number3
Publication statusPublished - Mar 2008
Externally publishedYes


  • Chloroplast
  • Electron paramagnetic resonance
  • Spin-probes
  • Transthylakoid proton gradient

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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