Effects of physical and chemical treatments on chloroplast manganese. NMR and ESR studies

Abstract

Measurements were made of the water proton relaxation rate ( T −1 2 = R 2), electron spin resonance (ESR) six-line signal of ‘free’ Mn 2+, and O 2-evolution activity in thylakoid membranes from pea leaves. The main results are: (1) Aging of thylakoids at 35°C causes a parallel decrease in O 2-evolution activity, in R 2 and in the content of bound Mn, suggesting that R 2 may be related to the loosely bound Mn involved in O 2 evolution. (2) Treatment of thylakoids with tetraphenylboron (TPB) at [TPB] > 2 mM produces a 2-fold increase in R 2, without release of Mn 2+. The titration curve exhibits three sharp end points. The first end point occurs at a [ TPB] [ chlorophyll] of 1.25, at which the O 2 evolution is completely inhibited. (3) Treatment of thylakoids with NH 2OH also increases R 2 by nearly 2-fold, either by the reduction of the higher oxidation states of Mn to Mn 2+ and / or by exposing the Mn to solvent protons. Also, progressive release of bound Mn occurs at [NH 2OH] ≥ 1 mM as shown by an increase increase in the Mn 2+ ESR signal and a decrease in R 2. (4) Addition of H 2O 2 (0.1–1.0%) to thylakoids causes an enhancement of R 2 similar to that by NH 2OH, but without the release of Mn 2+. (5) Heat treatment of thylakoids at 40–50°C releases Mn 2+ and increases R 2. Conversely, pH values of 7 to 4 release Mn 2+ without changing R 2 while pH values of 7–9 increase R 2 without releasing Mn 2+. Thus, both high and low pH values as well as the heat treatment cause structural changes enhancing the relaxivity of the bound Mn or of other paramagnetic species.