76 Biochimica et Biophysica Acta, 1059(1991) 76-90 ©A99i Elsevier Science Publishers B.V. 0005-2728/91/$03.50 ADONIS 000527289100181F BBABIO 43439 Stark effect spectroscopy of carotenoids in photosynthetic antenna and reaction center complexes David S. Gottfried, Martin A. Steffen and Steven G. Boxer Department of Chemistry, Stanford University, Stanford, CA (U.S.A.) (Received 10 December 1990) Key words: Stark effect; Electric field effect; Carotenoid; Carotenoid bandshift The effects of electric fields on the absorption spectra of the carotenoids spheroidene and spheroidenone in photosynthetic antenna and reaction center complexes (wild-type and several mutants) from purple non-sulfur bacteria are compared with those for the isolated pigments in organic glasses. In general, the field effects are substantially larger for the carotenoid in the protein complexes than for the extracted pigments and larger for spheroidenone than spheroidene. Furthermore, the electrochromic effects for carotenoids in all complexes are much larger than those for the Qx transitions of the bacteriochlorophyll and bacteriopheophytin pigments which absorb in the 450-700 nm spectral region. The underlying mechanism responsible for the Stark effect spectra in the complexes is found to be dominated by a change in permanent dipole moment of the carotenoid upon excitation. The magnitude of this dipole moment change is found to be considerably larger in the B800-850 complex compared to the reaction center for spheroidene; it is approximately equivalent in the two complexes for spheroidenone. These results are discussed in terms of the effects of differences in the carotenoid functional groups, isomers and perturbations on the electronic structure from interactions with the organized environment in the proteins. These data provide a quantitative basis for the analysis of carotenoid bandshifts which are used to measure transmem- brane potential, and they highlight some of the pitfalls in making such measurements on complex membranes containing multiple populations of carotenoids. The results for spheroidenone should be useful for studies of mutant proteins, since mutant strains are often grown semi-aerobically to minimize reversion. Introduction Carotenoids are widely distributed in nature and serve a wide range of functions [1]. They are especially important in photosynthetic systems, where they serve the dual functions of light harvesting and photoprotec- tion [2]. In addition to these important physiological roles, shifts in the absorption spectra of carotenoids have been widely used to measure transmembrane potentials and the electrogenicity of charge separation steps [3]. Underlying the utility of these band shifts is quantitative information on the change in dipole mo- Abbreviations: BChl, bacteriochlorophyll; LDS, lithium dodecyl sul- fate; PVA, poly(vinylalcohol); PMMA, poly(methylmethacrylate); 3MP, 3-methylpentane. Correspondence: S.G. Boxer, Department of Chemistry, Stanford University, Stanford, CA 94305, U.S.A. ment, A/~ A, and polarizability, Aot, for these chro- mophores in the protein environment. During the past few years, our laboratory and others have investigated the effects of applied electric fields (Stark effect spec- troscopy or electrochromism) on the absorption [4-7] and emission [8,9] spectra of photosynthetic reaction center (RC) complexes; Stark effect data for the bac- teriochlorophyll a (BChl a) Qy region of antenna complexes is presented in an accompanying paper [10]. In the course of these studies we examined the Qx region of the Stark effect spectrum for the R26 carotenoidless mutant of Rb. sphaeroides [4,5] and found only weak electrochromic effects. In the follow- ing we demonstrate that, when carotenoids are present in the protein complexes, much larger effects are ob- served in this spectral region, and these can be at- tributed to the carotenoid chromophores. Even larger effects are observed in antenna complexes. A prelimi- nary report focusing exclusively on spheroidene in the B800-850 antenna complex has been presented else-