Effect of manganese on the nuclear magnetic relaxivity of water protons in chloroplast suspensions.

Received February 4,198O SUMMARY Field dispersion profiles of the proton spin-lattice relaxation rate, ~1-1, in chloroplast suspensions show a local maximum near 20 MHz, probably due to bound Mn(I1); EDTA extraction eliminates, and MnC12 addition restores, the paramagnetic relaxivity. Since neither treatment affects water oxidation, the Mn(I1) site monitored appears to lie outside the water-splitting enzyme. Intense illumination almost totally suppresses the paramagnetic relaxivity through an electron-transport-dependent mechanism. Previous reports that chloroplast nuclear magnetic relaxivity varies cyclically in flash experiments require reevaluation in terms of the probable role of Mn(I1) that is nonfunctional in water oxidation. INTRODUCTION The relaxivity of the water proton resonance in concentrated suspensions of broken (Class II) chloroplasts has been reported to contain a large paramagnetic contribution (l-5). This component of the spin-lattice relaxation rate, Rl S l/Tl, shows a local maximum in the dispersion profile (plot of Rl against Larmor frequency) near 20 MHz, which is suggestive of membranebound Mn(I1) (1). Correlations of Rl with oxygen evolution activity and chloroplast manganese content were also reported and interpreted as arising from manganese in the water-splitting complex(3). Flash oscillations have been observed in the spin-spin relaxation rate and interpreted in terms ofoxidation state changes of manganese, acting as an intermediate in the water-splitting reaction(2,4,5). The measurements reported in this communication suggest that functional manganese does not contribute significantly to Rl in dark-adapted chloroplast suspensions. Endogenous nonfunctional Mn(I1) is a potent relaxing agent that probably accounts for previously observed effects in both dark-adapted and flash-illuminated chloroplasts. MATERIALS AND METHODS Broken chloroplasts were prepared from spinach as previously described(61, except that for some experiments, 1 mM EDTA was included in the homogenization buffer; KCN/Hg-inhibited chloroplasts were prepared by the method of Yocum and Guikema(7). The techniques for assay of 02 evolution and photophosphorylation activity have been reported previously(6). The standard buffer used for chloroplast suspensionandstoragewas 0.4Msucrose, 20 mM Tricine (pH 8) containing 15 mM NaCl. Samples (200~1) were illuminated where ABBREVIATIONS nmr, nuclear magnetic resonance; DCMU, 3-(3,4-dichlorophenyl)l,l-dimethylurea; PS, photosystem.