CHLOROPLAST THYLAKOID PROTEIN PHOSPHATASE REACTIONS ARE REDOX-INDEPENDENT AND KINETICALLY HETEROGENEOUS

At least eleven thylakoid proteins become phosphorylated under reducin g conditions, and redox titration has identified a common midpoint pot ential of E(m) = +38 +/- 4 mV, n = 0.95 +/- 0.06. In the presence of t he phosphatase inhibitor NaF (10 mM), the redox dependency of phosphor ylation is found to be essentially unchanged: E(m) = +50 +/- 3 mV, n = 1.02 +/- 0.04. Thylakoid membranes were phosphorylated in the light a nd then incubated at various redox potentials for 15 min in the dark; no redox dependency was observed in the dephosphorylation of any of th e 17 bands then distinguishable by autoradiography and phosphorimaging . The phosphoprotein phosphatase reactions can be divided arbitrarily into four kinetic classes: the fastest, class I, includes LHC II; the moderate class II includes D1 and D2; the slow class III includes CP43 and the 9 kDa phosphoprotein; finally, a 19.5 kDa protein exhibited n o loss of P-32 at all. In separate experiments we measured thylakoid p rotein dephosphorylation initiated by changing the redox potential fro m -140 to +200 mV, in the presence or absence of fluoride. In this cas e the results are consistent with at least two kinetically distinguish able classes of phosphoprotein phosphatase reactions. We conclude that thylakoid protein phosphatase reactions are kinetically heterogeneous and redox-independent. It follows that the redox dependency of thylak oid protein phosphorylation is a property of thylakoid protein kinase reactions. Our observed E(m) and n values are consistent with a primar y site of kinase redox control at the level of PQ/PQ.-of the Q(i) (Q(n )) site of the cytochrome b6/f complex.