Assembly of light-harvesting chlorophyll a/b complex in vitro. Time-resolved fluorescence measurements

The assembly kinetics of a pigment-binding membrane protein, the light-harvesting chlorophyll a/b complex of green plants, have been determined in vitro. Time-resolved fluorescence spectroscopy, with millisecond time resolution, has been used to monitor changes in both protein and chlorophyll (Chi) fluorescence, as well as in energy transfer from Chi b to Chi a, during complex assembly. Three reaction steps could be resolved after rapid, stopped-flow, mixing of the apoprotein (light-harvesting Chi a/b protein, LHCP) and pigments, solubilized in sodium dodecyl sulfate (SDS) and octyl glucoside (OG), respectively. A fast step in the range of 10 ms was detected regardless of whether the reaction mixture contained pigments or protein, or both, and is interpreted as being connected with the formation of mixed SDS and OG detergent micelles. Two further Steps were resolved: one with a time constant of about 1 min and another, slow step with a time constant of several minutes. Both of these were dependent on the presence of protein, Chls, and xanthophylls. Most, if not all, of the energy transfer from Chi b to Chi a was established during the slow step, indicating that the juxtaposition of these pigments, either by a structural rearrangement of the complex or by additional pigment binding, is the final stage in LHCII assembly in vitro.