Spin-probes designed for measuring the intrathylakoid pH in chloroplasts.
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 approximately 5.4-5.7 in the state of photosynthetic control, and pHin approximately 5.7-6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference DeltapH approximately 1.8-2.1. These values of DeltapH are consistent with a point of view that under steady-state conditions the proton gradient DeltapH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H+/ATP is n> or =4-4.7.
Tikhonov
Alexander N
AN
Department of Biophysics, Faculty of Physics, M.V. Lomonosov Moscow State University, Moscow, 119992, Russia. an_tikhonov@mail.ru
Agafonov
Roman V
RV
Grigor'ev
Igor A
IA
Kirilyuk
Igor A
IA
Ptushenko
Vasilii V
VV
Trubitsin
Boris V
BV
D000255:Adenosine Triphosphate / Q000378:metabolism; D002138:Calibration; D002736:Chloroplasts / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D003497:Cyclic N-Oxides / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods* / Q000592:standards; D006863:Hydrogen-Ion Concentration; D048289:Imidazolidines / Q000737:chemistry*; D048288:Imidazolines / Q000737:chemistry*; D008954:Models, Biological; D008956:Models, Chemical; D010070:Oxalates / Q000494:pharmacology; D010788:Photosynthesis; D017494:Proton Pumps / Q000378:metabolism; D018892:Proton-Motive Force; D013113:Spin Labels; D020524:Thylakoids / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D031307:Vicia faba / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects
D003497:Cyclic N-Oxides; D048289:Imidazolidines; D048288:Imidazolines; D010070:Oxalates; D017494:Proton Pumps; D013113:Spin Labels; D000255:Adenosine Triphosphate