R. W. Visschers

Energy transfer in the B800–850 antenna complex of purple bacteria Rhodobacter sphaeroides: A study by spectral hole-burning

Abstract The energy-transfer process within the isolated B800–850 pigment-protein complex of the purple bacterium Rhodobacter sphaeroides has been studied by means of spectral hole-burning. The band at 800 nm is inhomogeneously broadened because holes could be burnt into it. The widths of these permanent holes are independent of wavelength and temperature between 1.2 and 30 K. The BChl 800→BChl 850 energy-transfer time deduced from these widths is 2.3±0.4 ps for the isolated complex, and also for chromatophores at 1.2 K.

Inhomogeneous spectral broadening of the B820 subunit form of LH1

Abstract Site-selected fluorescence spectra of the B820 subunit of LH1 from Rhodospirillum rubrum G9 were measured at temperatures between 160 K and 4.2 K. A linear correlation between excitation wavelength and maximal emission wavelength was observed across the whole Q y absorption band of the B820 subunits, which persists at higher temperatures. This demonstrates that (a) the Q y absorbance band of the B820 subunit is inhomogeneously broadened; (b) the subunit consists of a strongly interacting dimer of BChl a ; (c) no energy transfer occurs among these dimers. The temperature dependence of the shape of the emission spectrum confirms that the protein forms a ‘glass-like’ environment for the bacteriochlorophyll pigments. After reassociating the octyl-glucoside-solubilized B820 subunits into the reassociated B873 form, the emission wavelength is independent of the wavelength of excitation at 4.2 K. This implies that the reassociated B873 form consists of a large number of interacting pigment molecules. The nature and origin of the spectral inhomogeneity in the B820 subunits is discussed.

Excited-state dynamics of mutated antenna complexes of purple bacteria studied by hole-burning

Absorption and fluorescence excitation spectra of various LH2 antenna complexes of two purple bacteria at low temperature (1.2 and 4.2 K) have been measured, and energy transfer rates within these complexes have been determined by spectra hole-burning. The systems studied were membranes of a wild-type strain of Rhodobacter sphaeroides , membrane samples from four LH2-only strains containing specifically mutated LH2 complexes of the same bacterium, and the isolated B800–820 complex of Rhodopseudomonas acidophila (strain 7050). The mutants exhibit blue-shifted B850 absorption bands with their spectral positions depending on the specific amino acid residues replaced in the α-polypeptide sequence. Energy transfer rates from B800 to B850 (or to their respectively blue-shifted bands) have been obtained by hole-burning experiments in the B800 band. The mutants of Rb. sphaeroides and the LH2 complex of Rps. acidophila yielded transfer times similar to those of the B800–850 complex of Rb. sphaeroides . These values, which for the various complexes vary between 1.7 and 2.5 ps in the wavelength region from 798 to 805 nm, do not decrease monotonically with the spectral distance between the bands. Various models based on Forsters energy transfer mechanism are discussed, of which only one is consistent with the results. In this model the energy is assumed to be transferred not directly from the Q y 0-0 band of B800 to that of the (blue-shifted) B850, but indirectly through the excitation of a vibrational mode.

Spectroscopic characterization of B820 subunits from light-harvesting complex I of Rhodospirillum rubrum and Rhodobacter sphaeroides prepared with the detergent n-octyl-rac-2,3-dipropylsulfoxide

Abstract B820 subunits from the light-harvesting complex 1 (LH1) were prepared by treatment of chromatophore membranes from Rhodospirillum rubrum with the detergent n -octyl- rac -2,3-dipropysulfoxide (ODPS). Similar subunits were obtained by dialysis of purified LH1 complexes from Rb. sphaeroides mutant M2192 against a buffer containing the same detergent. In contrast to other methods, no prior extraction of carotenoids is necessary for the preparation of these subunits when using the detergent ODPS. Reassociation of the B820(ODPS) subunits resulted in an apparently native, but carotenoid depleted LH1 complex. The presence of a fourth spectral component, with an absorbance maximum at 845 nm, was demonstrated by fitting the absorbance difference spectra with a sum of gaussians. Results from absorbance, fluorescence polarization, circular dichroism and triplet-singlet spectroscopy indicate that the subunits from LH1 obtained by this method are very similar to the B820 subunit isolated from carotenoid depleted light-harvesting complex 1 by octyl glucoside titration. These results imply that the B820 subunit structure created by detergent treatment is inherent to the structure of the functional LH1 antenna both for Rs. rubrum and Rb. sphaeroides .

Functional LH1 antenna complexes influence electron transfer in bacterial photosynthetic reaction centers

The effect of the light harvesting 1 (LH1) antenna complex on the driving force for light-driven electron transfer in the Rhodobacter sphaeroides reaction center has been examined. Equilibrium redox titrations show that the presence of the LH1 antenna complex influences the free energy change for the primary electron transfer reaction through an effect on the reduction potential of the primary donor. A lowering of the redox potential of the primary donor due to the presence of the core antenna is consistently observed in a series of reaction center mutants in which the reduction potential of the primary donor was varied over a 130 mV range. Estimates of the magnitude of the change in driving force for charge separation from time-resolved delayed fluorescence measurements in the mutant reaction centers suggest that the mutations exert their effect on the driving force largely through an influence on the redox properties of the primary donor. The results demonstrate that the energetics of light-driven electron transfer in reaction centers are sensitive to the environment of the complex, and provide indirect evidence that the kinetics of electron transfer are modulated by the presence of the LH1 antenna complexes that surround the reaction center in the natural membrane.

Spectral hole burning in pigment-protein complexes of photosynthetic bacteria

Abstract The excited-state dynamics of the peripheral light-harvesting complex LH2 (B800-850) of Rb. sphaeroides is compared to that of the dimer subunit (B8200(OG) of the core antenna complex LH1 of Rs. rubrum G9 at liquid helium temperatures by means of spectral hole burning. Both the B800 band of the LH2 complex and the absorption band of the B820(GO) subunit are inhomogeneously broadened. The homogeneous line width, Γ hom , of B800 is determined by energy transfer within the LH2 complex (√ transfer ~~0.8 to 2.5 ps); it is temperature independent between 1.2 and 30K and a factor of about 60 larger than that of B820(OG). By contrast, for B820(OG)Γ hom is only ∼ 1 GHz at 1.2 K; it depends on temperature and is predominatly given by dephasing.

Spectroscopic properties of pigment-protein complexes from photosynthetic purple bacteria in relation to their structure and function

In photosynthesis the light-energy, necessary to drive the electrochemical processes occurring in the reaction center, is collected by a light-harvesting antenna. Many (bacterio)chlorophyll-protein complexes cooperate in the absorption of the energy from the sun and the transport of the excitation energy to the reaction center.

Spectroscopic Characterization of a Subunit Form of Light Harvesting Complex I from Rhodospirillum rubrum and Rhodobacter sphaeroides

The Light Harvesting Antenna of photosynthetic purple bacteria consists of two major types: an inner core (or LH1) antenna, located in the vicinity of of the reaction center, and an outer peripheral antenna (or LH2) surrounding the core antenna. The core antenna complexes usually have an absorbance maximum around 880 nm and are produced in a fixed stochiometry to the photochemical reaction center. The core antenna complexes can be readily extracted from the photosynthetic membranes [1].