Different preparations of D1-D2-Cyt b559 complexes from spinach with different beta-carotene (Car) content [on average from <0.5 to 2 per reaction center (RC)] were studied by means of laser-induced optoacoustic spectroscopy. phiP680(+)Pheo(-) does not depend on the preparation (or on the Car content) inasmuch as the magnitude of the prompt heat (produced within 20 ns) does not vary for the different samples upon excitation at 675 and 620 nm. The energy level of the primary charge-separated state, P680(+)Pheo(-), was determined as EP680(+)Pheo(-) = 1.55 eV. Thus, an enthalpy change accompanying charge separation from excited P680 of deltaH*P680Pheo-->P680(+)Pheo(-) = -0.27 eV is obtained. Calculations using the heat evolved during the time-resolved decay of P680(+)Pheo(-) (< or = 100 ns) affords a triplet (3[P680Pheo]) quantum yield phi3[P680Pheo] = 0.5 +/- 0.14. The structural volume change, deltaV1, corresponding to the formation of P680(+)Pheo(-), strongly depends on the Car content; it is ca. -2.5 A3 molecule(-1) for samples with <0.5 Car on average, decreases (in absolute value) to -0.5 +/- 0.2 A3 for samples with an average of 1 Car, and remains the same for samples with two Cars per RC. This suggests that the Car molecules induce changes in the ground-state RC conformation, an idea which was confirmed by preferential excitation of Car with blue light, which produced different carotene triplet lifetimes in samples with 2 Car compared to those containing less carotene. We conclude that the two beta-carotenes are not structurally equivalent. Upon blue-light excitation (480 nm, preferential carotene absorption) the fraction of energy stored is ca. 60% for the 9Chl-2Car sample, whereas it is 40% for the preparations with one or less Cars on average, indicating different paths of energy distribution after Car excitation in these RCs with remaining chlorophyll antennae.