Manganese in photosynthetic oxygen evolution. I. Electron paramagnetic resonance study of the environment of manganese in Tris-washed chloroplasts

Abstract

A manganese electron paramagnetic resonance (EPR) signal not present in untreated chloroplasts is observed in chloroplasts treated by washing in Tris buffer (0.8 M, pH 8.0) for 15 min. Centrifugation indicates that the Mn responsible for the EPR signal is localized in the chloroplasts, not free in the supernatant liquid. Atomic absorption analysis demonstrates that less than 10% of total chloroplast Mn is lost upon Tris treatment. Tris treatment converts 60% of the total chloroplast Mn pool to an EPR-detectable state. Sonication causes the Mn EPR signal to be divided proportionately between the chloroplast pellet and the supernatant liquid. The EPR spectrum of Mn in Tris-washed chloroplasts is identical with that of a divalent aqueous Mn spectrum in g factor, hyperfine splitting, and linewidth temperature dependence. It is concluded that upon Tris treatment, Mn is released into the interior space of the thylakoid membrane. Transport of Mn and anionic chelating agents across the thylakoid membrane was investigated using EPR. The rate of Mn diffusion through the thylakoid membrane is slow, with a t 1 2 of 2.5 h. The rate of transfer of chelating agents such as EDTA is much faster, with t 1 2 of 750 ms. Tris washing also destroys a weak Mn-binding site on the exterior of the thylakoid membrane.