The state of detergent solubilised light-harvesting chlorophyll-a/b protein complex as monitored by picosecond time-resolved fluorescence and circular dichroism

Steady-state and picosecond time-resolved fluorescence techniques in conjunction with circular dichroism have been used to study the light-harvesting chlorophyll-a/b protein complex (LHC) isolated from pea chloroplasts. In particular, the effect of changing the detergent / chlorophyll ratio on the state of the LHC has been investigated. Our results have been interpreted in light of the known protein geometry of the LHC in 2-dimensional crystals (Kuhlbrandt, W. (1984) Nature 307, 478–479). The fluorescence lifetime data reveals 1 / e-lifetimes of 3.53 (±0.04) ns and 1.10 (±0.01) ns for a stable, efficiently energy-transferring state of the LHC. Subnanosecond lifetimes are observed under conditions leading to aggregation, while a long component of 5.50 (±0.16) ns corresponding to free Chl a is found when the detergent / chlorophyll ratio is high. The circular dichroism shows a major Chl-b exciton, a Chl-a / b exciton and a further ‘quenching’ Chl-b exciton. These have been attributed to: a C3 symmetric Chl-b interaction for which the intact C3 protein trimer geometry is a prerequisite; a dimeric Chl-a / b interaction, the presence of which is critically dependent on the detergent type; and a further Chl-b interaction which arises from the presence of aggregated trimers, respectively. We have found that the degree of heterogeneity with respect to the oligomeric state of the pigment-protein trimers is dependent upon the detergent / chlorophyll ratio used. Low detergent / chlorophyll ratios result in extensive aggregation of the trimers with a geometry similar to that found in 2-dimensional crystals of the LHC. Moderate detergent conditions yield predominantly non-aggregated trimers. Excess detergent conditions result in considerable chromophore heterogeneity and loss of the main Chl-b exciton consistent with protein denaturation through an initial break up of the trimer geometry. From these results we believe that in vitro the minimum stable functional unit corresponds to a C3 symmetric protein trimer.