Chloroplast thylakoid membranes isolated in the presence of EDTA retain high rates of 0 evolution (>340 #mol-hl'-mg chlorophyll'l) but contain no Mn1+ that is detectable by electron paramagnetic resonance (EPR) at room temperature, The total Mn2+ content of these preparations is 4.6 per 400 chlorophylls; 0.6 Mn2+ can be released by addition of Ca2 , a treatment that does not affect 02 evolution.The remaining Mn2+ (4 per 400 chlorophylls) appears to be functionally associated with 02 evolution activity. Inhibition by Tris, NH20H, or heat will release a small fraction of Mn2+ from these membranes (=25% with Tris, for example). Addition of Ca2+ further enhances Mn2+ release so that for Tris and for NH20H, 2 and 3, respectively, Mn2 per 400 chlorophylls are extracted from the 02-evolving complex. Based on the microwave power-saturation properties of the EPR MATERIALS AND METHODSThylakoid membranes were isolated from market spinach by a variety ofprocedures. Salt/EDTA membranes were isolated as described (5). Sucrose/MgCl2 and sucrose/EDTA chloroplasts were prepared by grinding leaves in sucrose buffer (0.4 M sucrose/20 mM Hepes, pH 7.5/15 mM NaCl)/2 mM MgCl2 or 1 mM EDTA. The pellets from these initial steps were washed with sucrose buffer/I mM EDTA and then with sucrose buffer/ 2 mM MgCl2 and then suspended in sucrose buffer. Finally, membranes were isolated by grinding leaves in sucrose buffer, pelleting the membranes, and suspending the pellet in sucrose buffer without further washing. The final suspensions [2.4-4.5 mg of chlorophyll (Chl)/ml] were either stored at -350C or used immediately. When assayed for activity, these preparations gave gramicidin-uncoupled rates of 02 evolution >300.mol-hr-'-mg Chl'-; one exception, the sucrose buffer-isolated and washed preparation, is below. The possibility that residual amounts of EDTA might contaminate these preparations was examined by addition of MnCl2 to the membrane suspensions before and after additional washes with 150 mM NaCl/4 mM MgCl2 or sucrose buffer. The levels of EPR-detectable Mn2+ found in these experiments indicated that the concentration of residual EDTA was <0.5 AuM. For Tris inactivation, a thylakoid suspension (0.6 ml) was mixed with 0.2 ml of 3.2 M Tris,-pH 8 (at 250C), allowed to stand in room light for 20 min at 30C, and then transferred to the EPR flat cell. When NH20H was the inactivating reagent, the suspension (0.6 ml) was mixed with 6Al of a 500 mM stock solution of NH20H in 0.01 M HC1 and then incubated in the dark for 20 min. Heat inactivation at 570Cwas carried out in an EPR flat cell as described (15). Inactivation of 02 evolution by these inhibitory treatments was assessed by the appearance of signal lIf or by direct assay of 02 evolution activity (or both).Abbreviation: Chl, chlorophyll. t To whom reprint requests should be addressed. 7507The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely t...

No abstract

No abstract

A new method of measuring the rate of the back reaction from the state Z+ P680 QA-in Tris-washed chloroplasts is described. By using ratios of back reaction rates we demonstrate a Tris-induced change in the equilibrium between Z end P680 and attribute this change to an alteration of the midpoint potential of Z by Tris treatment. We also demonstrate that the previously observed inhibition of the back reaction by ADRY reagents can be localized at Z and understood in terms of electron donation to Z' by ADRY reagents. Tris-washed chloroplasts Photosystem II EPR signal II_ ADR Y reagentBack reaction pH effect