EFFECTS OF PHYSICAL AND CHEMICAL TREATMENTS ON CHLOROPLAST MANGANESE NMR AND ESR STUDIES

Measurements were made of the water proton relaxation rate (T z- 1 = R 2), electron spin resonance (ESR) six-line signal of 'free ' Mn z+, and Oz-evolution activity in thylakoid membranes from pea leaves. The main results are: (1) Aging of thylakoids at 35°C causes a parallel decrease in Oz-evolution activity, in R z and in the content of bound Mn, suggesting that R z may be related to the loosely bound Mn involved in O z evolution. (2) Treatment of thylakoids with tetraphenyiboron (TPB) at [TPB]> 2 mM produces a 2-fold increase in R2, without release of Mn z+. The titration curve exhibits three sharp end points. The first end point occurs at a [TPBl/[chlorophyil] of 1.25, at which the 02 evolution is completely inhibited. (3) Treatment of thylakoids with NH2OH also increases R z 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 INH2OH]> 1 mM as shown by an increase in the Mn z+ ESR signal and a decrease in R 2. (4) Addition of H 2Oz (0.1-1.0%) to thylakoids causes an enhancement of R 2 similar to that by NHzOH, but without the release of Mn 2+. (5) Heat treatment of thylakoids at 40-50°C releases Mn z+ and increases R z. Conversely, pH values of 7 to 4 release Mn z+ without changing R 2 while pH values of 7-9 increase R 2 without releasing Mn z +. 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