CI-NMR measurement of chloride binding to the oxygen-evolving complex of spinach Photosystem II

The mechanism by which CIactivates the oxygen-evolving complex (OEC) of Photosystem II (PS II) in spinach was studied by 3SCI-NMR spectroscopy and steady-state measurements of oxygen evolution. Measurements of the excess 35Cl-NMR linewidth in dark-adapted, CI--depleted thylakoid and Photosystem I1 membranes show an overall hyperbolic decrease which is interrupted by sharp increases in linewidth (linewidth maxima) at approx. 0.3 mM, 0.75 mM, 3.25 mM (2.0 mM in PS II membranes), and 7.0 mM CI -. The rate of the Hill reaction ( H 2 0 ~ 2,6-dichlorophenolindophenol) at low light intensities (5% of saturation) as a function of [C! ] in thylakoids shows three intermediary plateaus in the concentration range between 0.1 and 10 mM CIindicating kinetic cooperativity with respect to CI-. The presence of linewidth maxima in the 35Cl-NMR binding curve indicates that CI addition exposes four types of CI binding site that were previously inaccessible to exchange with CIin the bulk solution. These results are best explained by proposing that C l binds to four sequestered (salt-bridged) domains within the oxygen-evolving complex. Binding of CI is facilitated by the presence of H + and vice versa. The pH dependence of the excess 3SCI-NMR linewidth at 0.75 mM CIshows that CIbinding has a maximum at pH 6.0 and two smaller maxima at pH 5.4 and 6.5 which may suggest that as many as three groups (perhaps histidine) with pK a values in the region may control the binding.