Reactivation of Photosynthetic Oxygen Evolution in Tris-inactivated Inside-out Photosystem Ii Vesicles from Spinach

Purified inside-out photosystem II vesicles with retained capacity for oxygen evolution and a simple polypeptide composition were found to be more sensitive to washing with tris(hydroxymethyl)aminomethane (Tris) at alkaline pH than right-side out thylakoids. This suggests that the thylakoid membrane limits the penetration of the active unprotonated form of Tris. The inhibition of photosynthetic oxygen evolution by alkaline Tris-washing (pH 8.5) coincided with the release of a polypeptide with a molecular weight of 32,000 from the inside-out vesicles. Only this polypeptide was released with concentrations of Tris up to 200 mM, while higher concentrations removed additional polypeptides with a molecular weight of 34,000 and 56,000. About 70 96 of the membrane bound manganese is lost by washing the vesicles with 200 mM-Tris. Oxygen evolution was not inhibited nor were polypeptides released with Tris buffered at pH 6.5. From right-side out thylakoids the 32,000 molecular weight polypeptide could not be extracted into the ambient medium by Tris-washing. Restoration of oxygen evolution of Tris-inactivated inside-out and right-side out vesicles was possible with the aid of reduced dichlorophenol indophenol but without the addition of the 32,000 molecular weight polypeptide or manganese.