Spin-probes designed for measuring the intrathylakoid pH in chloroplastsстатья
Статья опубликована в высокорейтинговом журнале
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Дата последнего поиска статьи во внешних источниках: 18 июля 2013 г.
Аннотация: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-diethy1-5,5-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 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) approximate to 5.4-5.7 in the state of photosynthetic control, and pH(in) approximate to 5.7-6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference Delta pH approximate to 1.8-2.1. These values of Delta pH are consistent with a point of view that under steady-state conditions the proton gradient Delta pH 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. (C) 2007 Elsevier B.V. All rights reserved.