An enzyme kinetics study of the pH dependence of chloride activation of oxygen evolution in photosystem II

Abstract

Oxygen evolution by photosystem II (PSII) involves activation by Cl^- ion, which is regulated by extrinsic subunits PsbQ and PsbP. In this study, the kinetics of chloride activation of oxygen evolution was studied in preparations of PSII depleted of the PsbQ and PsbP subunits (NaCl-washed and Na₂SO₄/pH 7.5-treated) over a pH range from 5.3 to 8.0. At low pH, activation by chloride was followed by inhibition at chloride concentrations >100 mM, whereas at high pH activation continued as the chloride concentration increased above 100 mM. Both activation and inhibition were more pronounced at lower pH, indicating that Cl^- binding depended on protonation events in each case. The simplest kinetic model that could account for the complete data set included binding of Cl^- at two sites, one for activation and one for inhibition, and four protonation steps. The intrinsic (pH-independent) dissociation constant for Cl^- activation, K _S, was found to be 0.9 ± 0.2 mM for both preparations, and three of the four pK _as were determined, with the fourth falling below the pH range studied. The intrinsic inhibition constant, K _I, was found to be 64 ± 2 and 103 ± 7 mM for the NaCl-washed and Na₂SO₄/pH7.5-treated preparations, respectively, and is considered in terms of the conditions likely to be present in the thylakoid lumen. This enzyme kinetics analysis provides a more complete characterization of chloride and pH dependence of O₂ evolution activity than has been previously presented.

Keywords: Chloride; Enzyme kinetics; Oxygen evolution; Photosystem II; Substrate inhibition; pH dependence.