STUDIES OF CA2-II USING CA-45(2+)( BINDING IN SPINACH PHOTOSYSTEM)

The Ca2+-binding properties of photosystem II were investigated with r adioactive Ca-45(2+). PS II membranes, isolated from spinach grown on a medium containing Ca-45(2+), contained 1.5 Ca2+ per PS II unit. Appr oximately half of the incorporated radioactivity was lost after incuba tion for 30 h in nonradioactive buffer. About 1 Ca2+/PS II bound slowl y to Ca2+-depleted membranes in the presence of the extrinsic 16- and 23-kDa polypeptides in parallel with restoration of oxygen-evolving ac tivity. The binding was heterogeneous with dissociation constants of 6 0 mu M (0.7 Ca2+/PS II) and 1.7 mM (0.3 Ca2+/PS II), respectively, whi ch could reflect different affinities of the dark-stable S-states for Ca2+. The reactivation of oxygen-evolving activity closely followed th e binding of Ca2+, showing that a single exchangeable Ca2+ per PS II i s sufficient for the water-splitting reaction to function. In PS II, d epleted of the 16- and 23-kDa polypeptides, about 0.7 exchangeable Ca2 +/PS II binds with a dissociation constant of 26 mu M, while 0.3 Ca2binds with a much weaker affinity (K-d > 0.5 mM). The rate of binding of Ca2+ in the absence of the two extrinsic polypeptides was significa ntly higher than with the polypeptides bound. The rate of dissociation of bound Ca2+ in the dark, which had a half-time of about 80 h in int act PS II, increased in the absence of the 16- and 23-kDa polypeptides and showed a further increase after the additional removal of the 33- kDa protein and manganese. The rate of dissociation was also significa ntly faster in weak light than in the dark regardless of the presence or absence of the 16- and 23-kDa polypeptides. Removal of the 33-kDa d onor-side polypeptide together with the two lighter ones led to a redu ction in the amount of bound Ca2+, while practically no Ca2+ bound aft er treatments to dissociate also the manganese of the water-oxidizing site.