This review describes the structures of the two major integral membrane pigment complexes, the RC-LH1 'core' and LH2 complexes, which together make up the light-harvesting system present in typical purple photosynthetic bacteria. The antenna complexes serve to absorb incident solar radiation and to transfer it to the reaction centres, where it is used to 'power' the photosynthetic redox reaction and ultimately leads to the synthesis of ATP. Our current understanding of the biosynthesis and assembly of the LH and RC complexes is described, with special emphasis on the roles of the newly described bacteriophytochromes. Using both the structural information and that obtained from a wide variety of biophysical techniques, the details of each of the different energy-transfer reactions that occur, between the absorption of a photon and the charge separation in the RC, are described. Special emphasis is given to show how the use of single-molecule spectroscopy has provided a more detailed understanding of the molecular mechanisms involved in the energy-transfer processes. We have tried, with the help of an Appendix, to make the details of the quantum mechanics that are required to appreciate these molecular mechanisms, accessible to mathematically illiterate biologists. The elegance of the purple bacterial light-harvesting system lies in the way in which it has cleverly exploited quantum mechanics.

Intense sunlight is dangerous for photosynthetic organisms. Cyanobacteria, like plants, protect themselves from light-induced stress by dissipating excess absorbed energy as heat. Recently, it was discovered that a soluble orange carotenoid protein, the OCP, is essential for this photoprotective mechanism. Here we show that the OCP is also a member of the family of photoactive proteins; it is a unique example of a photoactive protein containing a carotenoid as the photoresponsive chromophore. Upon illumination with blue-green light, the OCP undergoes a reversible transformation from its dark stable orange form to a red ''active'' form. The red form is essential for the induction of the photoprotective mechanism. The illumination induces structural changes affecting both the carotenoid and the protein. Thus, the OCP is a photoactive protein that senses light intensity and triggers photoprotection.cyanobacteria ͉ nonphotochemical quenching ͉ photoprotection ͉ phycobilisome

Spirilloxanthin and spheroidene were reconstituted into the carotenoidless B850 light-harvesting (LH) complex from the Rhodobacter (Rb.) sphaeroides R-26.1 mutant with the aim to obtain new insights in energy transfer, triplet formation, and other relaxation phenomena in photosynthetic light harvesting. Resonance Raman measurements showed that spirilloxanthin and spheroidene are bound to the B850 complex in the same planar configuration, whereas spirilloxanthin in its native LH1 complex of Rhodospirillum (Rs.) rubrum assumes a twisted configuration. Ultrafast transient absorption measurements with excitation of the carotenoid molecules to their S2 state enabled us to identify, in both reconstituted B850 complexes, the recently found S* carotenoid singlet excited state and the direct generation of carotenoid triplet states within picoseconds through the singlet fission mechanism [Gradinaru, C. C., et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 2364−2369]. Global analysis has allowed us to quantify the formation yields of these states. In the B850 complex reconstituted with spheroidene, the triplet yield is 5−10%, similar to that found on the spheroidene-binding LH2 complex of Rb. sphaeroides 2.4.1 [Papagiannakis, E. et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6017−6022]. The triplet state of spirilloxanthin in the B850 complex is formed with a similar low yield, in contrast to the native LH1 complex of Rs. rubrum where the triplet yield is as large as 25−30%. This illustrates that the formation of the triplet state depends on the type of complex that binds the carotenoid and not on the carotenoid itself. More specifically, the singlet fission process that underlies ultrafast triplet formation is more efficient when spirilloxanthin is bound in a distorted configuration (in Rs. rubrum) than with either spirilloxanthin or spheroidene bound in a planar configuration (in Rb. sphaeroides). The extent of geometrical deformation of the carotenoid imposed by binding to the LH complexes partly determines the carotenoid light-harvesting function by either deactivating the excited-state energy of S* by transformation into a triplet pair or allowing this energy to flow to bacteriochlorophyll. Comparison of the energy transfer properties in the spheroidene-reconstituted B850 complex, which lacks the B800 bacteriochlorophyll, with that of the LH2 complex of Rb. sphaeroides 2.4.1 suggests that, apart from a light-harvesting function, the B800 bacteriochlorophylls in LH2 may have an important role in funneling the photon energy absorbed by carotenoids toward the reaction center.

Predicting the complete electronic structure of carotenoid molecules remains an extremely complex problem, particularly in anisotropic media such as proteins. In this paper, we address the electronic properties of nine relatively simple carotenoids by the combined use of electronic absorption and resonance Raman spectroscopies. Linear carotenoids exhibit an excellent correlation between (i) the inverse of their conjugation chain length N, (ii) the energy of their S0 → S2 electronic transition, and (iii) the position of their ν1 Raman band (corresponding to the stretching mode of their conjugated C═C bonds). For cyclic carotenoids such as β-carotene, this correlation is also observed between the latter two parameters (S0 → S2 energy and ν1 frequency), whereas their "nominal" conjugation length N does not follow the same relationship. We conclude that β-carotene and cyclic carotenoids in general exhibit a shorter effective conjugation length than that expected from their chemical structure. In addition, the effect of solvent polarizability on these molecular parameters was investigated for four of the carotenoids used in this study. We demonstrate that resonance Raman spectroscopy can discriminate between the different effects underlying shifts in the S0 → S2 transition of carotenoid molecules.

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Resonance Raman spectroscopy was performed on peripheral light-harvesting proteins from Rhodobacter sphaeroides in which the residue betaArg-10 has been modified by site-selected mutagenesis. We show that this residue is indeed involved (as proposed by X-ray crystallographic studies on the LH2 complex from Rhodopseudomonas acidophila), in an H-bond with the acetyl carbonyl of the 800 nm-absorbing BChl in these proteins (B800), and that the presence of such an H-bond induces a ca. 10 nm red shift of the lowest energy transition (Qy) of this molecule. Moreover, other parameters involved in the fine tuning of the absorption of the B800 molecules may be determined from our experiments, and we propose that the local electromagnetic properties of the B800 binding site may induce an additional 10 nm red shift of this transition. These results constitute the first experimental evidence for the parameters able to modify in vivo the absorption of "monomeric" BChl molecules, i.e. BChl not involved in strong excitonic interactions, and will be of great help for understanding the absorption properties of such pigments in other light-harvesting systems.

The photosynthetic light-harvesting systems of purple bacteria and plants both utilize specific carotenoids as quenchers of the harmful (bacterio)chlorophyll triplet states via triplet-triplet energy transfer. Here, we explore how the binding of carotenoids to the different types of light-harvesting proteins found in plants and purple bacteria provides adaptation in this vital photoprotective function. We show that the creation of the carotenoid triplet states in the light-harvesting complexes may occur without detectable conformational changes, in contrast to that found for carotenoids in solution. However, in plant light-harvesting complexes, the triplet wavefunction is shared between the carotenoids and their adjacent chlorophylls. This is not observed for the antenna proteins of purple bacteria, where the triplet is virtually fully located on the carotenoid molecule. These results explain the faster triplet-triplet transfer times in plant light-harvesting complexes. We show that this molecular mechanism, which spreads the location of the triplet wavefunction through the pigments of plant light-harvesting complexes, results in the absence of any detectable chlorophyll triplet in these complexes upon excitation, and we propose that it emerged as a photoprotective adaptation during the evolution of oxygenic photosynthesis.

We have characterized the influence of the protein environment on the spectral properties of the bacteriochlorophyll (Bchl) molecules of the peripheral light-harvesting (or LH2) complex from Rhodobacter sphaeroides. The spectral density functions of the pigments responsible for the 800 and 850 nm electronic transitions were determined from the temperature dependence of the Bchl absorption spectra in different environments (detergent micelles and native membranes). The spectral density function is virtually independent of the hydrophobic support that the protein experiences. The reorganization energy for the B850 Bchls is 220 cm(-1), which is almost twice that of the B800 Bchls, and its Huang-Rhys factor reaches 8.4. Around the transition point temperature, and at higher temperatures, both the static spectral inhomogeneity and the resonance interactions become temperature-dependent. The inhomogeneous distribution function of the transitions exhibits less temperature dependence when LH2 is embedded in membranes, suggesting that the lipid phase protects the protein. However, the temperature dependence of the fluorescence spectra of LH2 cannot be fitted using the same parameters determined from the analysis of the absorption spectra. Correct fitting requires the lowest exciton states to be additionally shifted to the red, suggesting the reorganization of the exciton spectrum.