Two-photon and fluorescence spectroscopy and the effect of environment on the photochemical properties of peridinin in solution and in the peridinin-chlorophyll-protein from amphidinium carterae

The ground and excited-state properties of peridinin in solution and in the peridinin-chlorophyll-protein (PCP) complex are studied by using one-photon and two-photon spectroscopy, solvent effects, and quantum theory. Two-photon excitation spectra, two-photon polarization data, and fluorescence spectra in CS₂ reveal three low-lying excited singlet states in peridinin: a lowest-excited¹Ag^*--like state with a system origin at ∼16 200 cm^-1, a ¹B_u^*+-like S₂ state with a system origin at ∼19 300 cm^-1, and a ¹B_u^*--like S₃ state with a system origin at ∼22 000 cm^-1. The ¹B_u^*+-like S₂ state dominates the two-photon excitation spectrum of peridinin in solution and in PCP because of type II enhancement associated with a large oscillator strength (f ≈ 1.6) coupled with a change in dipole moment upon excitation (Δμ ≈ 3 D). Thus, the two-photon spectrum looks very much like the one-photon spectrum although weak vibronic bands of the ¹A_g^*--like state are observed at ∼17, ∼18.1, and ∼19.2 kK in the two-photon spectrum of peridinin in CS₂. MNDO-PSDCI theory and solvent effect studies indicate that the S₁ (¹A_g^*--like) state has a large dipole moment (μ_aa ≈ 16 D, Δμ ≈ 8 D) in both polar and nonpolar environments, much larger than the ground-state dipole moment [μ₀₀ ≈ 6 (non polar media) - 8 (polar media) D]. Thus, the ¹A_g^*--like state is assigned as the charge-transfer state observed in previous studies. The suggestion that the charge-transfer character is induced in polar solvent is not supported by these studies. We conclude that some of the studies that have suggested an increase in the charge-transfer character of S₁ with solvent polarity are based on experiments that are more sensitive to (μ_aa - μ₀₀) μ₀₀ than (μ_aa - μ₀₀). Peridinin exists as a mixture of all-trans and 14-s-cis (single bond to allene moiety) conformers in ambient temperature hexane solution, with a predominance of the former. Polar solvents such as methanol and high-dielectric solvents such as CS₂ preferentially stabilize the all-trans conformer relative to the 14-s-cis. MNDO-PSDCI calculations on the minimized peridinin molecules within PCP indicate that most of the chromophores have excited state properties similar to those observed for the isolated all-trans chromophore. However, the chromophores occupying sites 612 and 622 are not only blue shifted but have inverted S₁ and S₂ singlet states. Our studies provide both support and additional perspective on the PCP energy transfer model proposed by Damjanovic et al. (Biophys. J. 2000, 79, 1695-1705) in which the peridinin molecules in these sites transfer energy to other peridinin chromophores rather than directly to chlorophyll. We conclude that the 612 and 622 sites optimize energy transfer by increasing the population of the lowest-excited ¹B_u^*+- like state, which provides enhanced dipolar coupling to the remaining peridinin set. An analysis of the PCP complex spectrum in terms of component spectra of the pigments indicates that two peridinin molecules have unique, blue-shifted spectra.