Jan Amesz

Biophysical techniques in photosynthesis

Preface. Part One: Optical Methods. 1. Developments in Classical Optical Spectroscopy J. Amesz. 2. Linear and Circular Dichroism G. Garab. 3. Fluorescence K. Sauer, M. Debreczeny. 4. Ultrafast Spectroscopy of Photosynthetic Systems R. Jimenez, G.R. Fleming. 5. Data Analysis of Time-Resolved Measurements A.R. Holzwarth. 6. Photosynthetic Thermoluminescence as a Simple Probe of Photosystem II Electron Transport Y. Inoue. 7. Accumulated Photon Echo Measurements of Excited State Dynamics in Pigment-Protein Complexes T.J. Aartsma, R.J.W. Louwe, P. Schellenberg. 8. Spectral Hole Burning: Methods and Applications to Photosynthesis N. Raja, S. Reddy, G.J. Small. 9. Infrared and Fourier-Transform Infrared Spectroscopy W. Mantele. 10. Resonance Raman Studies in Photosynthesis - Chlorophyll and Carotenoid Molecules B. Robert. 11. Stark Spectroscopy of Photosynthetic Systems S.G. Boxer. 12. The Photoacoustic Method in Photosynthesis - Monitoring and Analysis of Phenomena which Lead to Pressure Changes Following Light Excitation S. Malkin. Part Two: Magnetic Resonance. 13. Magnetic Resonance: an Introduction A.J. Hoff. 14. Time-Resolved Electron Paramagnetic Resonance Spectroscopy - Principles and Applications H. Levanon. 15. Electron Spin Echo Methods in Photosynthesis Research R.D. Britt. 16. ENDOR Spectroscopy W. Lubitz, F. Lendzian. 17. Optically Detected Magnetic Resonance (ODMR) of Triplet States in Photosynthesis A.J. Hoff. 18. MagicAngle Spinning Nuclear Magnetic Resonance of Photosynthetic Components H.J.M. de Groot. Part Three: Structure and Oxygen. 19. Structure Determination of Proteins by X-Ray Diffraction M. Schiffer. 20. Electron Microscopy E.J. Boekema, M. Rogner. 21. X-Ray Absorption Spectroscopy:Determination of Transition Metal Site Structures in Photosynthesis V.K. Yachandra, M.P. Klein. 22. Mossbauer Spectroscopy P.G. Debrunner. 23. Characterization of Photosynthetic Supramolecular Assemblies Using Small Angle Neutron Scattering D.M. Tiede, P. Thiyagarajan. 24. Measurements of Photosynthetic Oxygen Evolution H.J. van Gorkom, P. Gast.

Pigment organization of the B800–850 antenna complex of Rhodopseudomonas sphaeroides

Abstract The B800–850 antenna complex of Rhodopseudomonas sphaeroides was studied by comparing the spectral properties of several different types of complexes, isolated from chromatophores by means of the detergents lithium dodecyl sulfate (LDS) or lauryl dimethylamine N -oxide (LDAO). Fluorescence polarization spectra of the BChl 800 emission at 4 K indicated that rapid energy transfer between at least two BChl 800 molecules occurs with a rate constant of energy transfer k ET > 3 · 10 12 s −1 . The maximal dipole-dipole distance between the two BChl 800 molecules was calculated to be 18–19 A. The porphyrin rings of the BChl 800 molecules are oriented parallel to each other, while their Q y transition moments are mutually perpendicular. The energy-transfer efficiency from carotenoid to bacteriochlorophyll measured in different complexes showed that two functionally different carotenoids are present associated with, respectively, BChl 800 and BChl 850. Fluorescence polarization and linear dichroism spectra revealed that these carotenoids have different absorption spectra and a different orientation with respect to the membrane. The carotenoid associated with BChl 800 absorbs some nanometers more to the red and its orientation is approximately parallel to the membrane, while the carotenoid associated with BChl 850 is oriented more or less perpendicular to the membrane. The fluorescence polarization of BChl 850 was the same for the different complexes. This indicates that the observed polarization of the fluorescence is determined by the smallest complex obtained which contains 8–10 BChl 850 molecules. The B800–850 complex isolated with LDAO thus must consist of a highly ordered array of smaller structures. On basis of these results a minimal model is proposed for the basic unit consisting of four BChl 850 and two BChl 800 and three carotenoid molecules.

Spectroscopic properties of the reaction center and of the 47 kDa chlorophyll protein of Photosystem II

The D1-D2-cytochrome b-559 reaction center complex and the 47 kDa antenna chlorophyll protein isolated from spinach Photosystem II were characterized by means of low temperature absorption and fluorescence spectroscopy. The low temperature absorption spectrum of the D1-D2-cytochrome b-559 complex showed two bands in the Qy region located at 670 and 680 nm. On the basis of its absorption maximum and orientation the latter component may be attributed at least in part to P-680, the primary electron donor of Photosystem II. The 47 kDa antenna complex showed absorption bands at 660, 668 and 677 nm and a minor component at 690 nm. The latter transition appeared to be associated with the characteristic low temperature 695 nm fluorescence band of Photosystem II. The 695 nm emission band was absent in the D1-D2 complex, which indicates that it does not originate from the reaction center pheophytin, as earlier proposed. The transition dipole responsible for the main fluorescence at 684 nm from this complex had a parallel orientation with respect to the membrane plane in the native structure. The reaction center preparation contains two spectrally distinct carotenoids with different orientations.

Low-temperature optical properties and pigment organization of the B875 light-harvesting bacteriochlorophyll-protein complex of purple photosynthetic bacteria

Abstract Optical and structural properties of the B875 light-harvesting complex of purple bacteria were examined by measurements of low-temperature circular dichroism (CD) and excitation spectra of fluorescence polarization. In the B875 complex isolated from wild-type Rhodopseudomonas sphaeroides, fluorescence polarization increased steeply across the long-wavelength Qy bacteriochlorophyll a (BChl) absorption band at both 4 and approx. 300 K. With the native complex in the photosynthetic membranes of Rhodospirillum rubrum and Rps. sphaeroides wild-type and R26-carotenoidless strains, this significant increase in polarization from 0.12 to 0.40 was only observed at low temperature. A polarization of −0.2 was observed upon excitation in the Qx BChl band. The results indicate that about 15% of the BChl molecules in the complex absorb at wavelengths about 12 nm longer than the other BChls. All BChls have approximately the same orientation with their Qy transition dipoles essentially parallel and their Qx transitions perpendicular to the plane of the membrane. At low temperature, energy transfer to the long-wavelength BChls is irreversible, yielding a high degree of polarization upon direct excitation, whereas at room temperature a partial depolarization of fluorescence by energy transfer between different subunits occurs in the membrane, but not in the isolated complex. CD spectra appear to reflect the two spectral forms of B875 BChl in Rps. sphaeroides membranes. They also reveal structural differences between the complexes of Rps. sphaeroides and Rhs. rubrum, in both BChl and carotenoid regions. The CD spectrum of isolated B875 indicates that the interactions between the BChls but not the carotenoids are altered upon isolation.

Spectroscopic properties of chloroplast grana membranes and of the core of photosystem ii

An oxygen-evolving Photosystem II core complex essentially free of the light-harvesting chlorophyll ab protein complex, containing 45 chlorophylls per reaction center was isolated from spinach chloroplasts. Its structural integrity was established by studying its photochemistry and spectral properties. The absorption spectrum measured at 4 K revealed the presence of at least five spectrally distinct chlorophyll a species. The same bands, but in different proportions, were observed in a Photosystem II grana preparation used as starting material for the preparation of the core complex. The relative contributions of these components to the overall absorption were calculated by deconvoluting this spectrum into Gaussian bands. The core complex was enriched in a long-wave band located at 683 nm, which presumably reflects the presence of 8–10 pigment molecules that are closely associated with the reaction center. Low temperature fluorescence emission spectra showed the characteristic Photosystem II emission bands located at 685 nm (F685) and at 695 nm (F685). The two states giving rise to these emissions are in thermal equilibrium down to 70 K. It is suggested that F685 arises from a chlorophyll a species absorbing at 676 nm and that F695 is the result of fluorescence from the photoactive pheophytin a absorbing around 683 nm.

Identification of 81-hydroxychlorophyll a as a functional reaction center pigment in heliobacteria

Chromatographic analysis of membranes and antenna-reaction center complexes of Heliobacterium chlorum and membranes of Heliobacillus mobilis revealed the presence of a small amount of a polar pigment with an absorption spectrum which was essentially identical to that of chlrophyll (Chl) a . Its structure was elucidated by means of 1 H-NMR Correlated Spectroscopy, 13 C-NMR and 252 Cf-Plasma Desorption Mass Spectrometry as 8 1 -hydroxychlorophyll a (8 1 -OH-Chl a ), esterified with farnesol. It is concluded that 8 1 -OH-Chl a is responsible for the band near 670 nm in the absorption spectrum of heliobacteria. The molar ratio bacteriochlorophyll (BChl) g : 8 1 -OH-Chl a was approx. 17 for rapidly growing cells of H. chlorum and Hb. mobilis . On the assumption that the antenna consists of 35–40 BChls g , this means that two molecules of 8 1 -OH-Chl a are present per reaction center. At least one of them functions as electron acceptor in the primary charge separation.

Bacteriochlorophyll g epimer as a possible reaction center component of heliobacteria

Normal-phase HPLC analysis of acetone extracts of cells, membranes and antenna-reaction center complexes of Heliobacterium chlorum and Heliobacillus mobilis showed the presence of bacteriochlorophyll (BChl) g′, the 132-epimer of BChl g. The molar ratio of BChl g:BChl g′ in these preparations was approx. 18 in cells and membranes of both species. This value, when combined with a molar ratio of BChl g to the primary electron donor, P-798, of 35–40, yields a BChl g′:P-798 ratio of 2, suggesting that P-798 may be a dimer of BChl g′. The amount of BChl g′ in the antenna-reaction center complex of H. chlorum was slightly higher, suggesting that some epimerization may have occurred during the isolation of the complex. In contrast, bacteriopheophytins (BPhe) g and g′ were present in too small amouts to be essential components of the photosynthetic apparatus. This confirms the idea that the reaction center of heliobacteria is basically different from those of purple bacteria and Photosystem II. A detailed interpretation, based on Correlated Spectroscopy and Double Resonance experiments is given of the 1H-NMR spectra of BChl g and BChl g′, confirming the structure and identity of both pigments. Absorption and circular dichroism spectra of BChl g and BChl g′ are also presented.

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus : II. The chlorosome.

The transfer of excitation energy and the pigment arrangement in isolated chlorosomes of the thermophilic green bacterium Chloroflexus aurantiacus were studied by means of absorption, fluorescence and linear dichroism spectroscopy, both at room temperature and at 4 K. The low temperature absorption spectrum shows bands of the main antenna pigments BChl c and carotenoid, in addition to which bands of BChl a are present at 798 and 613 nm. Fluorescence measurements showed that excitation energy from BChl c and carotenoid is transferred to BChl a, which presumably functions as an intermediate in energy transfer from the chlorosome to the cytoplasmic membrane. Measurements of fluorescence polarization and the use of two different orientation techniques for linear dichroism experiments enabled us to determine the orientation of several transition dipole moments with respect to each other and to the three principal axes of the chlorosome. The Qy transition of BChl a is oriented almost perfectly perpendicular to the long axis of the chlorosome. The Qy transition of BChl c and the γ-carotene transition dipole are almost parallel to each other. They make an angle of about 40° with the long axis and of about 70° with the short axis of the chlorosome; the angle between these transitions and the BChl a Qy transition is close to the magic angle (55°).

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus

We have studied the pigment arrangement in purified cytoplasmic membranes of the thermophilic green bacterium Chloroflexus aurantiacus. The membranes contain 30–35 antenna bacteriochlorophyll a molecules per reaction center; these are organized in the B808–866 light-harvesting complex, together with carotenoids in a 2:1 molar ratio. Measurements of linear dichroism in a pressed polyacrylamide gel permitted the accurate determination of the orientation of the optical transition dipole moments with respect to the membrane plane. Combination of linear dichroism and low temperature fluorescence polarization data shows that the Qy transitions of the BChl 866 molecules all lie almost perfectly parallel to the membrane plane, but have no preferred orientation within the plane. The BChl 808 Qy transitions make an average angle of about 44° with this plane. This demonstrates that there are clear structural differences between the B808–866 complex of C. aurantiacus and the B800–850 complex of purple bacteria. Excitation energy transfer from carotenoid to BChl a proceeds with about 40% efficiency, while the efficiency of energy transfer from BChl 808 to BChl 866 approaches 100%. From the minimal energy transfer rate between the two spectral forms of BChl a, obtained by analysis of low temperature fluorescence emission spectra, a maximal distance between BChl 808 and BChl 866 of 23 Å was derived.

Excited states and primary charge separation in the pigment system of the green photosynthetic bacterium Prosthecochloris aestuarii as studied by picosecond absorbance difference spectroscopy

Abstract Picosecond absorbance difference spectra at a number of delay times after a 35 ps excitation flash and kinetics of absorbance changes were measured of the membrane vesicle preparation Complex I from the photosynthetic green sulfur bacterium Prosthecochloris aestuarii . After chemical oxidation of the primary donor the excitation pulse produced singlet and triplet excited states of carotenoid and bacteriochlorophyll a . With active reaction centers present also the flash-induced primary charge separation and subsequent electron transfer were observed. The singlet excited state of the carotenoid, formed by direct excitation at 532 nm, is characterized by an absorbance band peaking at 590 nm. Its average lifetime was calculated to be about 1 ps. Excited singlet states of bacteriochlorophyll a were characterized by a bleaching of their ground state Q y absorption bands. Singlet excited states, localized on the so-called core complex, were produced by energy transfer from excited carotenoid. Their lifetime was about 70 ps. A decay component of about 280 ps was ascribed to singlet excited bacteriochlorophyll a in the bacteriochlorophyll a protein. These singlet excitations were partly converted to the triplet state. With active reaction centers, oxidation of the primary donor, P-840, characterized by the bleaching of its Q y and Q x absorption bands, was observed. This oxidation was accompanied by a bleaching between 650 and 680 nm and an absorbance increase between 680 and 750 nm. These changes, presumably due to reduction of bacteriopheophytin c (Van Bochove, A.C., Swarthoff, T., Kingma, H., Hof, R.M., Van Grondelle, R., Duysens, L.N.M. and Amesz, J. (1984) Biochim. Biophys. Acta 764, 343–346), were attributed to the reduction of the primary electron acceptor. Electron transfer to a secondary acceptor occurred with a time-constant of 550 ± 50 ps. Since no absorbance changes due to reduction of this acceptor were observed in the red or infrared region, we tentatively assume that this acceptor is an iron-sulfur center.