Oxygen evolution in the absence of the 33-kilodalton manganese-stabilizing protein

Abstract

There has been a considerable amount of controversy concerning the ability of photosystem II to evolve oxygen in the absence of the 33-kDa, manganese-stabilizing protein. Early reports indicated that some capacity for oxygen evolution existed in manganese-stabilizing protein-depleted membranes while more recent studies have suggested that the observed oxygen evolution activity arose from residual manganese-stabilizing protein present in the salt-washed preparations. In this paper, it is conclusively demonstrated that significant rates of steady-state oxygen evolution are observed in oxygen-evolving photosystem II membranes in the absence of detectable quantities of the manganese-stabilizing protein. More then 99% of the manganese-stabilizing protein was removed by either one CaCl2 or two NaCl-urea washes. The amount of manganese-stabilizing protein removed was quantified immunologically using mouse polyclonal antibodies. Oxygen evolution rates of 115-140 mumol of O2 (mg of Chl)-1 h-1 were observed in the NaCl-urea-washed preparations. These rates represent about 24% of the rate observed in untreated membranes [450-600 mumol of O2 (mg of Chl)-1 h-1]. Somewhat lower, although still significant rates were observed in the CaCl2-washed preparations. Optimal rates of oxygen-evolving activity in NaCl-urea-washed membranes which are devoid of the manganese-stabilizing protein required high concentrations of calcium and chloride.

An Intrinsically Disordered Photosystem II Subunit, PsbO, Provides a Structural Template and a Sensor of the Hydrogen-bonding Network in Photosynthetic Water Oxidation

“…The use of 7 mM potassium ferricyanide alone, as in the FT-IR experiments, gave a steady-state oxygen evolution rate of 250 Ϯ 45 mol of O 2 (mg of Chl-h) Ϫ1 . Depletion of Extrinsic Subunits-PSII samples were incubated in high ionic strength buffer (400 mM sucrose, 50 mM MES-NaOH (pH 6.0), and 2 M NaCl) to extract the extrinsic polypeptides PsbP and PsbQ (NaCl-PSII) (77,79,80). To remove PsbO, NaCl-PSII was incubated in buffer containing 400 mM sucrose, 50 mM MES-NaOH (pH 6.0), 2.6 M urea, and 200 mM NaCl (Urea-PSII) (77,79,80).…”

“…Depletion of Extrinsic Subunits-PSII samples were incubated in high ionic strength buffer (400 mM sucrose, 50 mM MES-NaOH (pH 6.0), and 2 M NaCl) to extract the extrinsic polypeptides PsbP and PsbQ (NaCl-PSII) (77,79,80). To remove PsbO, NaCl-PSII was incubated in buffer containing 400 mM sucrose, 50 mM MES-NaOH (pH 6.0), 2.6 M urea, and 200 mM NaCl (Urea-PSII) (77,79,80). As an alternative method for PsbO removal, NaCl-PSII was incubated in buffer containing 400 mM sucrose, 50 mM MES-NaOH (pH 6.0), and 1 M CaCl 2 (CaCl 2 -PSII) (79).…”

“…To remove PsbO, NaCl-PSII was incubated in buffer containing 400 mM sucrose, 50 mM MES-NaOH (pH 6.0), 2.6 M urea, and 200 mM NaCl (Urea-PSII) (77,79,80). As an alternative method for PsbO removal, NaCl-PSII was incubated in buffer containing 400 mM sucrose, 50 mM MES-NaOH (pH 6.0), and 1 M CaCl 2 (CaCl 2 -PSII) (79).…”

“…A Western blot containing a standard curve of NaCl-PSII samples (data not shown) was employed (as performed previously in Ref. 79). Comparison of the integrated areas showed that urea or CaCl 2 treatments removed 82 and 92% of native PsbO, respectively.…”

Deprotonation of the 33-kDa, Extrinsic, Manganese-stabilizing Subunit Accompanies Photooxidation of Manganese in Photosystem II

“…In the presence of low concentrations of calcium and chloride, plant MSP was found to be required for photosynthetic oxygen evolution and for maintaining the stability of the manganese cluster (9,10). In the presence of high concentrations of calcium and chloride, oxygen evolution occurs, but the steady state rate of enzymatic activity is impaired upon removal of MSP (7,9,(11)(12)(13)(14). In addition, removal of MSP and replacement with calcium and chloride result in kinetic inhibition of the S state transitions (12,(15)(16)(17)(18)(19).…”