Abstract
Calcium and chloride are activators of oxygen evolution in photosystem II (PSII), the light-absorbing water oxidase of higher plants, algae, and cyanobacteria. Calcium is an essential part of the catalytic Mn(4)CaO(5) cluster that carries out water oxidation and chloride has two nearby binding sites, one of which is associated with a major water channel. The co-activation of oxygen evolution by the two ions is examined in higher plant PSII lacking the extrinsic PsbP and PsbQ subunits using a bisubstrate enzyme kinetics approach. Analysis of three different preparations at pH 6.3 indicates that the Michaelis constant, K(M), for each ion is less than the dissociation constant, K(S), and that the affinity of PSII for Ca(2+) is about ten-fold greater than for Cl(-), in agreement with previous studies. Results are consistent with a sequential binding model in which either ion can bind first and each promotes the activation by the second ion. At pH 5.5, similar results are found, except with a higher affinity for Cl(-) and lower affinity for Ca(2+). Observation of the slow-decaying Tyr Z radical, Y(Z)•, at 77 K and the coupled S(2)Y(Z)• radical at 10 K, which are both associated with Ca(2+) depletion, shows that Cl(-) is necessary for their observation. Given the order of electron and proton transfer events, this indicates that chloride is required to reach the S(3) state preceding Ca(2+) loss and possibly for stabilization of Y(Z)• after it forms. Interdependence through hydrogen bonding is considered in the context of the water environment that intervenes between Cl(-) at the Cl-1 site and the Ca(2+)/Tyr Z region.