Topology of NH2OH-induced Mn(II) release from chloroplast thylakoid membranes

Abstract

Permeant and impermeant metal ion chelators have been used in conjunction with NMR relaxation time measurements (T1) of solvent protons to probe the membrane topology of the Mn(II) released from the water-oxidizing center of chloroplast thylakoid membranes by NH2OH. Chelex, a tightly binding divalent metal ion exchanger, quantitatively removes Mn2+ (added as MnCl2) from the external thylakoid membrane without significantly affecting oxygen evolution activity or photophosphorylation efficiency. Because of its obvious impermeance (the resin is supplied as 0.2 mm beads), chelex selectively removes only manganese that is in equilibrium with the external aqueous phase. Both internal and external manganese pools are removed by chelex in the presence of A23187, a divalent cation-specific ionophore. Topological experiments using these reagents have shown that NH2OH releases Mn(II) predominantly to the loculus in freshly prepared, dark-adapted thylakoid membranes at 0-3[deg]C. This topology changes radically as a result of three pretreatments: (1) incubation of thylakoid membranes in the dark at 25[deg]C, which redirects Mn(II) release toward the external medium with a half-time of 10-15 min; (2) illumination with saturating white light, which decreases the half-time of reorientation to about 1 min; (3) freeze-thawing in 0.4 M sucrose, which results in the appearance of 40-60% of the NH2OH-liberated Mn(II) in the external medium. None of these treatments substantially degrades O2 evolution activity or osmotic integrity as judged from measurements of photophosphorylation efficiency. It is concluded that the topology of the manganese site associated with photosystem II is not static but changes dramatically in response to external stimuli, possibly reflecting a regulatory mechanism of photophosphorylation.