J. Phys. Chem. B 1999, 103, 2279-2290
2279
Temperature Dependence of Optical Spectra of Bacteriochlorophyll a in Solution and in Low-Temperature Glasses Emanuele Bellacchio and Kenneth Sauer* Department of Chemistry, UniVersity of California and Physical Biosciences DiVision, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ReceiVed: September 3, 1998; In Final Form: December 7, 1998
Absorption, fluorescence emission, and fluorescence excitation spectra of bacteriochlorophyll a [BChl a] are examined throughout the temperature range from 298 to 79 K in several glass-forming solvents. Changes in the absorption spectra that occur continuously throughout this range may reflect increased extent of coordination of the central Mg, changes in solvent dielectric, and/or altered hydrogen bonding. Fluorescence emission spectra exhibit a new feature that grows steadily, beginning at temperatures below about 250 K in solvents that are hydrogen-bond donors: 1-propanol and 2-propanol. The emerging fluorescence band, located about 300 cm-1 to the blue of the fluorescence band seen at higher temperatures, achieves nearly equal amplitude at 163 K and below. It is noteworthy that no corresponding feature appears in the absorption on the blue side of the Qy absorption band. The Kennard-Stepanov relation between absorption and fluorescence, which holds with somewhat elevated T* values in the high-temperature region, is seen to fail dramatically at lower temperatures as the short-wavelength fluorescence feature grows. The short-wavelength feature is interpreted as fluorescence resulting from an excited electronic state that is conformationally unrelaxed. At temperatures below 178 K evidence for additional spectroscopic features appears, especially in conjunction with measurements of emission spectra using different excitation wavelengths and of excitation spectra of fluorescence measured at different emission wavelengths. This is in the region of matrix glass formation, and the new BChl a components may reflect site inhomogeneity. Similar spectroscopic studies of BChl a in non-hydrogen-bonding solvents do not provide evidence of new blue-shifted fluorescence in the 298-79 K temperature range. They do, however, exhibit evidence of site inhomogeneity in the low-temperature glass matrices. Implications are discussed regarding the interpretation of low-temperature spectral features that have been reported for photosynthetic membranes and isolated pigment-proteins.
I. Introduction The spectroscopic properties of chlorophyll pigments are strikingly sensitive to molecular environment and interchromophore interactions.1-4 This is particularly true for bacteriochlorophyll a (BChl a) both in vivo and in vitro.2,5-8 In the membranes of photosynthetic bacteria the BChl a molecules in both the reaction centers and the light-harvesting complexes show multiple spectroscopic forms, particularly in the longwavelength (Qy band) absorption region. In vitro studies have shown that the wavelength, intensity, and band shape of BChl a are dependent on factors such as solvent polarity,9,10 temperature,11 extent of ligation of the central magnesium,12 and BChl aggregation.13 Although all of the principal transitions (Qy, Qx, Bx, and By, in order of increasing energy) respond to solvent polarity, the Qx and By transitions are reported to be especially sensitive to Mg coordination,12 and the Qy region is especially sensitive to BChl a aggregation or associations that result in excitonic coupling between molecules.14 Many spectroscopically distinct forms of chlorophyll pigments have been described on the basis of studies of intact biological materials or of pigment-proteins, etc. derived from them. Among the most interesting of these are long-wavelength pigment bands that are seen best upon cooling samples to low * To whom correspondence should be addressed. Mailing address: Melvin Calvin Laboratory, University of California, Berkeley, 94720-5230. Fax: (510) 486-6059. E-mail:
[email protected].
temperature.15 These species were often first identified from their unusually strong fluorescence, which grows progressively with decreasing temperature. Associated absorption features can sometimes be seen, often as a shoulder or incompletely resolved peak on the low-energy side of the main Qy absorption band.16 In many cases the energy of the long-wavelength component seen in low-temperature spectra is lower than that of the reaction center, and concern has been raised about how excitation into these low-energy bands results in primary photochemistry, which, at least in some cases, remains highly efficient even at 10 K and below.17 The participation, or even the presence, of these low-energy components at room temperature is less well documented. However, there are organisms, such as Rhodopseudomonas Viridis and Heliobacterium chlorum, where the major long-wavelength antenna pigment component absorbs wavelengths longer than those characteristic of the corresponding reaction center; nevertheless, these organisms carry out active photosynthesis at room temperature and exhibit efficient excitation transfer from the antenna to the reaction center even at 10 K.17 At room temperature the greater thermal energy available increases the possibility of thermally assisted excitation trapping mechanisms, but the low-temperature observations present a quandary that is currently unresolved. To explore further the molecular origin of these spectroscopic effects, we have undertaken a study of the temperature dependence of the spectroscopy of BChl a in solution, especially
10.1021/jp9836123 CCC: $18.00 © 1999 American Chemical Society Published on Web 03/09/1999
2280 J. Phys. Chem. B, Vol. 103, No. 12, 1999 in solvents that form clear glasses below their melting temperatures. We restricted our studies to conditions where pigment aggregation is negligiblesspecifically, low concentrations and solvents that are at least somewhat polar. A straightforward interpretation of the spectra of porphyrins, including BChl a, can be made using the four-orbital model developed by Gouterman.18 These are delocalized π- and π*-type orbitals associated with the conjugated porphyrin macrocycle. The Qy and Bx transitions arise from excitation of electrons from the HOMO to the LUMO and the LUMO + 1, respectively. The Qx and By transitions are attributed to electrons excited from the HOMO - 1 to the LUMO and LUMO + 1, respectively. Variations in optical spectra of chlorophyll a (Chl a), BChl a, and related molecules in dilute solutions owing to different solvents or changing temperature have been attributed to one or more of the following factors: (1) the effect of solvent polarity differentially on the energy of the orbitals,10,19,20 (2) the extent of coordination to the central Mg atom,20-23 which particularly affects the energy of the HOMO - 1, (3) influences of solvent polarity and hydrogen bonding on carbonyl substituents that are more or less conjugated with the π-electron system of the porphyrin.20,24,25 In the case of BChl a, the effects mentioned above have been documented in a number of studies.12 The effect of changing Mg ligation from five-coordinate to six-coordinate using solvents of greater polarity (electron donation ability), for example, results in shifts to long wavelength of the Qx and By transitions owing to the effect on the HOMO - 1.26 Similar spectroscopic effects that occur as a function of temperature in a single solvent system and that had been reported previously11 were interpreted by Evans and Katz12 from analysis of IR spectra to result from changes in Mg coordination. The Qy and Bx transition energies are much less affected, which implicates the energy of the HOMO - 1 as the origin of this distinctive effect of BChl Mg ligation. Detailed molecular orbital calculations by Petke et al.27 support the conclusion from the earlier four-orbital model of Gouterman18 that the HOMO - 1 exhibits high electron density at the bacteriochlorin nitrogen atoms that interact directly with the central Mg involved in ligand binding. The extent of Mg coordination has been established using NMR and vibrational spectroscopy, especially IR and resonance Raman. Solvent dielectric (permittivity) also contributes to these changes in spectroscopic properties owing to influences on the bacteriochlorin macrocycle and on the conjugated carbonyls. These influences can be seen through the effects of solvent dielectric on the visible absorption band positions and on the frequencies of vibrational modes that have significant macrocycle and/or carbonyl involvement.28 In all of these studies, tetrahydroporphyrins such as BChl a have a distinct advantage for the spectroscopist because of the nearly complete separation of the Qy, Qx and Soret (overlapping Bx and By) spectral regions. II. Materials and Methods Bacteriochlorophyll a. The method of Omata and Murata29 was used to isolate BChl a from the purple, non-sulfur bacterium Rhodobacter sphaeroides. This method involves chromatography on Sepharose CL-6B (Fluka). The final chromatographic fractions containing the separated BChl a were collected in glass tubes and the solvents evaporated under flowing N2. All procedures of pigment separation and purification were carried out at approximately 0 °C and in darkness. The BChl a was stored as the solid in a freezer. Solvents. 2-Methyltetrahydrofuran containing 1% BHT, butylated hydroxytoluene [2,6-di-(tert-butyl)-4-methylphenol], 1-pro-
Bellacchio and Sauer panol, 2-propanol, hexane, and 1,4-dioxane were obtained from Sigma. Pyridine was obtained from Fisher and triethylamine from Fluka. Solvents were all of the highest purity commercially available and were used without further purification. Spectroscopy. The preparation of solutions for absorption and fluorescence measurements was carried out in darkness. The BChl a concentrations used for fluorescence provided an absorbance of 0.01 at the Qy maximum for a 1 cm path. This corresponds to a concentration of
