Studies on the rate-limiting reaction of photosynthetic oxygen evolution in spinach chloroplasts

The modulated oxygen polarography has been used to study the rate-determining steps of photosynthetic oxygen evolution in spinach chloroplasts. The rate constant, k, of the reaction has a value of 218 +/- 10 (S.E.) s-1 at 23 degrees C and an activation energy of 7 +/- 2 (S.E.) kcal-mol-1. A kinetic isotope experiment indicated that this step is probably not the water-splitting reaction. These findings resemble previous results with the unicellular alga Chlorella (Sinclair, J. and Arnason, T (1974) Biochim. Biophys. Acta 368, 393-400). In other experiments we changed the pH, O2 concentration and osmolarity of the medium, and treated the chloroplasts with 1 mM NH4Cl without detecting any significant change in k. These results suggest that the step is irreversible. However, a significantly lower value of k, 110 +/- 20 (S.E.) s-1 was obtained when all salts except 1 mM MgCl2 were removed from the medium bathing the chloroplasts.