A Mechanism of Energy Dissipation in Cyanobacteria

When grown under a variety of stress conditions, cyanobacteria express the isiA gene, which encodes the IsiA pigment-protein complex. Overexpression of the isiA gene under iron-depletion stress conditions leads to the formation of large IsiA aggregates, which display remarkably short fluorescence lifetimes and thus a strong capacity to dissipate energy. In this work we investigate the underlying molecular mechanism responsible for chlorophyll fluorescence quenching. Femtosecond transient absorption spectroscopy allowed us to follow the process of energy dissipation in real time. The light energy harvested by chlorophyll pigments migrated within the system and eventually reaches a quenching site where the energy is transferred to a carotenoid-excited state, which dissipates it by decaying to the ground state. We compare these findings with those obtained for the main light-harvesting complex in green plants (light-harvesting complex II) and artificial light-harvesting antennas, and conclude that all of these systems show the same mechanism of energy dissipation, i.e., one or more carotenoids act as energy dissipators by accepting energy via low-lying singlet-excited S₁ states and dissipating it as heat.     

Trapping, loss and annihilation of excitations in a photosynthetic system: II. Experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata

In this paper a number of experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata is described in which the total fluorescence yield and/or the total fraction of reaction centers closed after a picosecond laser pulse were measured as a function of the pulse intensity. The conditions were such that the reaction centers were either all in the open or all in the closed state before the pulse arrived. These experiments are analysed using the theoretical formalism discussed in the preceding paper (Den Hollander, W.T.F., Bakker J.G.C., and Van Grondelle, R., Biochim. Biophys. Acta 725, 492–507). From the experimental results the number of connected photosynthetic units, λ, the rate of energy transfer between neighboring antenna molecules, k_h, and the rate of trapping by an open reaction center, k^o_t, can be estimated. For R. rubrum it is found that λ = 14−17, k_h = (1−2)·10¹² s⁻¹ and k^o_t = (4−6)·10¹¹ s⁻¹, for Rps. capsulata λ ≈ 30, k_h ≈ 4·10¹¹ s⁻¹ and k^o_t ≈ 3·10¹¹ s⁻¹. The findings are discussed in terms of current models for the structure of the antenna and the kinetic properties of the decay processes occurring in these purple bacteria.     

Complex between single-stranded DNA and gene 5 protein of bacteriophage M13 studied with linear dichroism and ultraviolet absorption

We have studied complexes between the gene 5 protein (gp5) of bacteriophage M13 and various polynucleotides, including single-stranded DNA, using ultraviolet absorption and linear dichroism. Upon complex formation the absorption spectra of both the protein and the polynucleotides change. The protein absorption changes indicate that for at least two of the five tyrosine residues per protein monomer the environment becomes less polar upon binding to the polynucleotides but also to the oligonucleotide p(dT)8. All gp5-polynucleotide complexes give rise to intense linear dichroism spectra. These spectra are dominated by negative contributions from the bases, but also a small positive dichroism of the protein can be discerned. The spectra can be explained by polynucleotide structures, which are the same in all complexes. The base orientations are characterized by a substantial inclination and propellor twist. The number of possible combinations of inclination and propeller twist values, which are in agreement with the linear dichroism results, is rather limited. The base orientations with respect to the complex axis are essentially different from those in the complex with the single-stranded DNA-binding protein gp32 of bacteriophage T4.     

Simultaneous measurement of electric birefringence and dichroism. A study on photosystem 1 particles.

We have developed a straightforward method to separate linear-dichroism and birefringence contributions to electric-field induced signals in a conventional birefringence setup. The method requires the measurement of electric birefringence for three different angular positions of the analyzer. It is demonstrated that the presence of linear dichroism can significantly influence the measured signals and lead to completely erroneous calculations of the birefringence signal and field-free decay times if its contribution is not taken into account. The new method is used to determine electric birefringence and linear dichroism of trimeric Photosystem 1 complexes from the cyanobacterium Synechocystis PCC 6803 in the detergents n-dodecyl-beta-D-maltoside and n-octyl-beta-D-glucoside. It is concluded that the orientation of the particles in the field is predominantly caused by a permanent electric dipole moment that is directed parallel to the symmetry axis of the particles. Comparison of the decay times obtained with dodecylmaltoside and octylglucoside supports a model in which the thickness of the disc-like complexes remains similar (7-8 nm) upon replacing dodecylmaltoside by octylglucoside, whereas the diameter increases from 14.4 +/- 0.2 to 16.6 +/- 0.2 nm because of an increased thickness of the detergent layer. This change in diameter is in good agreement with electron-microscopy results on Photosystem 2 complexes in dodecylmaltoside and octylglucoside (Dekker, J. P., E. J. Boekema, H. T. Witt, and M. Rögner. 1988. Biochim. Biophys. Acta 936:307-318). The value of approximately 16.6 nm for the diameter of Photosystem 1 trimers in dodecylmaltoside is in good agreement with recent results obtained from electron microscopy in combination with extensive image analysis (Kruip, J., E. J. Boekema, D. Bald, A. F. Boonstra, and M. Rögner. 1993. J. Biol. Chem. 268:23353-23360).     

Polarized fluorescence and absorption of macroscopically aligned Light Harvesting Complex II.

Polarized absorption and fluorescence measurements have been performed at 77 K on isotropic and anisotropic preparations of trimeric Light Harvesting Complex II (LHC-II) from spinach. The results enable a decomposition of the absorption spectrum into components parallel and perpendicular to the trimeric plane. For the first time, it is shown quantitatively that the strong absorption band around 676 nm is polarized essentially parallel to the plane of the trimer, i.e., the average angle between the corresponding transition dipole moments and this plane is at most 12 degrees. The different absorption bands for LHC-II should not be considered as corresponding to individual pigments but to collective excitations of different pigments. Nevertheless, the average angle between the Qy transition dipole moments of all chlorophyll a pigments in LHC-II and the trimeric plane could be determined and was found to be 17.5 degrees +/- 2.5 degrees. For the chlorophyll b pigments, this angle is significantly larger (close to 35 degrees). At 77 K, most of the fluorescence stems from a weak band above 676 nm and the corresponding transition dipole moments are oriented further out of plane than the dipole moments corresponding to the 676-nm band. The results are shown to be of crucial significance for understanding the relation between the LHC-II structure and its spectroscopy.     

A study of the primary charge separation in green bacteria by means of flash spectroscopy

A reaction-center pigment-protein complex of the green bacterium Prosthecochloris aestuarii was studied by means of nanosecond-flash spectroscopy. In this complex electron transfer between the primary and secondary acceptor is blocked. The spectra and kinetics of the absorption changes induced by a short flash indicated the formation of the radical pair P-840⁺I^?, which decayed in 20–35 ns, mainly to the triplet state of the primary electron donor P-840. The absorption difference spectrum of the initial absorption change indicated that the primary acceptor I is either bacteriopheophytin c or another pigment with absorption maximum at 665 nm.     

On the quenching of the fluorescence yield in photosynthetic systems

A modified matrix model describing transfer of excitation energy in the photosynthetic pigment system is discussed. In addition to the antenna pigments and reaction centers of the simple matrix model, a coupling complex is postulated mediating energy transfer between antenna and reaction centers. The values of the parameters describing the transfer properties of the coupling complex can be chosen in such a way that a number of recent unexplained measurements of fluorescence properties of various purple bacteria can be described. If such coupling complexes are present in oxygen evolving organisms, some of their properties must be different from those of purple bacteria.     

Non-random conformation of a mouse IgG2a monoclonal antibody at low pH

The pH dependence of the conformation of a mouse IgG2a, kappa monoclonal antibody (MN12) was investigated by several physical techniques, including fluorescence spectroscopy, near-ultraviolet and far-ultraviolet CD, and electric-field-induced transient birefringence measurements. The intensity of the intrinsic tryptophan fluorescence remained constant in the pH range from 3.5 to 10.0. A conformational alteration in the MN12 molecule was observed in the pH region between pH 3.5 and 2.5, as reflected by a substantial enhancement of the fluorescence quantum yield. This effect was more pronounced at high ionic strengths. The fluorescence emission was unaltered, indicating that the acid-induced conformational state is different from a completely unfolded state. This was confirmed by CD and fluorescence polarisation measurements. Iodide and acrylamide fluorescence quenching studies indicated a gradually increasing accessibility of MN12 tryptophan residues with decreasing pH. At low pH precipitation was observed in the presence of iodide. One rotational relaxation time (0.16-0.18 microseconds) was observed for MN12 by electric-field-induced transient birefringence measurements at low ionic strength. After exposure of MN12 to low pH for 1 h, the relaxation time was increased to 0.23 microseconds; a further increase to 0.30 microseconds was observed after 24 h. The combined results suggest an acid-induced expansion and enhanced flexibility of MN12, which eventually leads to irreversible aggregation.     

Alpha-crystallin exists in a non-spherical form. A study on the rotational properties of native and reconstituted alpha-crystallin.

Native alpha-crystallin, obtained from the cortex of calf lenses with FPLC (Pharmacia) was characterized by means of transient-electric-birefringence measurements and ultraviolet linear-dichroism spectroscopy. These techniques were also performed on 6-M-urea-dissociated and reconstituted alpha-crystallin. Transient-electric-birefringence measurements offer the possibility to characterize the often observed, but usually neglected, non-spherical occurrences of alpha-crystallin in more detail. Although not distinguishable with size-exclusion chromatography, we could identify at least two different classes of both native and reconstituted alpha-crystallin, from which at least one consists of non-spherical molecules. The results are compared with those obtained with electron microscopy using different staining methods. From the three independent techniques used we find evidence that a fraction of the alpha-crystallin exists in a more extended quaternary structure. The results are difficult to explain with a concentric three-layer model for alpha-crystallin as proposed by Tardieu et al. [Tardieu, A., Laporte, D., Licinio, P., Krop, B. & Delaye, M. (1986) J. Mol. Biol. 192, 711-724].     

Fluorescence polarization and low-temperature absorption spectroscopy of a subunit form of light-harvesting complex I from purple photosynthetic bacteria

Measurements of polarized fluorescence and CD were made on light-harvesting complex 1 and a subunit form of this complex from Rhodospirillum rubrum, Rhodobacter sphaeroides, and Rhodobacter capsulatus. The subunit form of LH1, characterized by a near-infrared absorbance band at approximately 820 nm, was obtained by titration of carotenoid-depleted LH1 complexes with the detergent n-octyl beta-D-glucopyranoside as reported by Miller et al. (1987) [Miller J. F., Hinchigeri, S. B., Parkes-Loach, P. S., Callahan, P. M., Sprinkle, J. R., & Loach, P. A. (1987) Biochemistry 26, 5055-5062]. Fluorescence polarization and CD measurements at 77 K suggest that this subunit form must consist of an interacting bacteriochlorophyll a dimer in all three bacterial species. A small, local decrease in the polarization of the fluorescence is observed upon excitation at the blue side of the absorption band of the B820 subunit. This decrease is ascribed to the presence of a high-energy exciton component, perpendicular to the main low-energy exciton component. From the extent of the depolarization, we estimate the oscillator strength of the high-energy component to be at most 3% of the main absorption band. The optical properties of B820 are best explained by a Bchl a dimer that has a parallel or antiparallel configuration with an angle between the Qy transition dipoles not larger than 33 degrees. The importance of this structure is emphasized by the results showing that core antennas from three different purple bacteria have a similar structure.(ABSTRACT TRUNCATED AT 250 WORDS)