35CI-NMR measurement of chloride binding to the oxygen-evolving complex of spinach Photosystem II

Abstract

The mechanism by which Cl − activates the oxygen-evolving complex (OEC) of Photosystem II (PS II) in spinach was studied by 35Cl-NMR spectroscopy and steady-state measurements of oxygen evolution. Measurements of the excess 35Cl-NMR linewidth in dark-adapted, Cl −-depleted thylakoid and Photosystem II 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 Cl −. The rate of the Hill reaction (H 2O → 2,6-dichlorophenolindophenol) at low light intensities (5% of saturation) as a function of [Cl −] in thylakoids shows three intermediary plateaus in the concentration range between 0.1 and 10 mM Cl − indicating kinetic cooperativity with respect to Cl −. The presence of linewidth maxima in the 35Cl-NMR binding curve indicates that Cl − addition exposes four types of Cl − binding site that were previously inaccessible to exchange with Cl − in the bulk solution. These results are best explained by proposing that Cl − binds to four sequestered (salt-bridged) domains within the oxygen-evolving complex. Binding of Cl − is facilitated by the presence of H + and vice versa. The pH dependence of the excess 35Cl-NMR linewidth at 0.75 mM Cl − shows that Cl − binding 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 p K a values in the region may control the binding.