Henk Vasmel

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.

Photoreduction of menaquinone in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus

Abstract Photochemically active reaction centers were isolated from the facultatively aerobic gliding green bacterium Chloroflexus aurantiacus . The absorption difference spectrum, obtained after a flash, reflected the oxidation of P-865, the primary donor, and agreed with that observed in a purified membrane preparation from the same organism (Bruce, B.D., Fuller, R.C. and Blankenship, R.E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6532–6536). By analysis of the kinetics in the presence of reduced N -methylphenazonium methosulfate to prevent accumulation of oxidized P-865, the absorption difference spectrum of an electron acceptor was obtained. The electron acceptor was identified as menaquinone (vitamin K-2), which is reduced to the semiquinone anion in a stoichiometry of approximately one molecule per reaction center. Reduction of menaquinone was accompanied by changes in pigment absorption in the infrared region. Our results indicate that the electron-acceptor chain of C. aurantiacus is very similar to that of purple bacteria.

Electron transport components of Heliobacterium chlorum investigated by EPR spectroscopy at 9 and 35 GHz

In membrane fragments of Heliobacterium chlorum we have observed by EPR spectroscopy the light‐induced reduction of a ferredoxin type electron acceptor with g y = 1.937 and g z = 1.879 at a midpoint potential E m > −420 mV. A low‐potential (E m < −620 mV) electron acceptor at g = 2.0038 having a 15 G wide EPR line at 9.2 GHz and 18 G at 35 GHz could also be reduced by photoaccumulation; it is either located in the electron transport chain or on a side path. The linewidth of the primary donor P‐798 shows an anomalous temperature dependence, possibly caused by a change in dimeric structure induced by cooling. A scheme for the proposed electron transport chain is presented.

Isolation and properties of a pigment-protein complex associated with the reaction center of the green photosynthetic sulfur bacterium Prosthecochloris aestuarii.

Abstract The membrane-bound pigment system of green sulfur bacteria consists of light-harvesting bacteriochlorophyll a -protein and a ‘core complex’ that is associated with the reaction center (Kramer, H.J.M., Kingma, H., Swarthoff, T. and Amesz, J. (1982) Biochim. Biophys. Acta 681, 359–364). The isolation and properties of the core complex from Prosthecochloris aestuarii are described. The complex has a molecular mass of 200 ± 50 kDa and contains bacteriochlorophyll a , carotenoid and pigments absorbing near 670 nm (probably bacteriopheophytin c and an unidentified pigment). Fluorescence emission spectra and sodium dodecyl sulfate polyacrylamide gel electrophoresis showed the absence of light-harvesting bacteriochlorophyll a -protein. The preparation showed no reaction center activity. Circular and linear dichroism spectra indicated that the structure of the core complex was basically not altered by the isolation procedure. Comparison with the CD spectrum of the intrinsic membrane-bound pigment-protein complex indicates that the latter contains 14 bacteriochlorophyll a molecules (two subunits) belonging to the light-harvesting protein and about 20 bacteriochlorophyll a molecules belonging to the core complex.

Analysis by exciton theory of the optical properties of the Chloroflexus aurantiacus reaction center

The optical properties of the reaction center of the filamentous green bacterium Chloroflexus aurantiacus, that contains three bacteriochlorophyll (BChl) a and three bacteriopheophytin (BPh) a molecules, were analyzed in the near-infrared region with the aid of exciton theory. The coordinates obtained from the X-ray analysis of the reaction center of Rhodopseudomonas viridis (Deisenhofer, J., Epp, O., Miki, K., Huber, R. and Michel, H. (1984) J. Mol. Biol. 180, 385–398) were used for the geometry of the reaction center of C. aurantiacus, with the replacement of one of the ‘accessory’ BChl molecules by BPh. The results were found to be in good agreement with experimental low-temperature absorption spectra, linear and circular dichroism and fluorescence polarization spectra and lead to the following conclusions. The allowed, low-energy exciton transition of the primary electron donor (P-865) is located at 887 nm and carries the dipole strength of approx. two BChl a monomers; the high-energy exciton transition, around 790 nm, is mixed with wave functions of other pigments, which explains its relatively small angle with respect to the 887 nm transition. The optical transition of the accessory BChl a molecule near 812 nm has some contribution of the BChls that constitute P-865. This can account for the experimentally observed reorientation and shift of this transition upon oxidation of P-865. Two of the BPh molecules are located on the same (probably the M) polypeptide subunit and show a clear splitting of absorption bands (11 nm) due to exciton coupling; the single BPh on the opposite branch shows hardly any exciton shift. Similar calculations for reaction centers of purple bacteria that contain four BChl a and two BPh a molecules resulted in a very low dipole strength for the high-energy transition of the primary donor due to antisymmetric mixing with both accessory BChl a wave functions and gave very little splitting of the absorption bands of BPh a. Our results indicate that the arrangement of the chromophores in reaction centers of C. aurantiacus is very similar to that in purple bacteria. The functional L-chains of the reaction centers of purple and filamentous green bacteria consist of pigments of the same type in a probably very similar arrangement.

Pigment composition of the photosynthetic membrane and reaction center of the green bacterium Prosthecochloris aestuarii

Abstract We have performed a quantitative analysis of the pigment composition of different pigment-protein complexes present in the membrane of the green sulfur bacterium Prosthecochloris aestuarii, using the resolving power of reversed-phase high-performance liquid chromatography. The most purified photochemically active complexes contained only carotenoids (OH-chlorobactene and rhodopin), bacteriochlorophyll a and a chlorophyllous pigment with absorption maxima at 663 and 433 nm, like bacteriochlorophyll c. However, the lipophilicity of this pigment, labeled BChl 663, is quite high and indicates that it contains 5–6 additional methylene groups compared to the BChl c homologue known as most lipophilic. Comparison of the BChl 663 content of various pigment-protein complexes indicates that BChl 663 is present in an amount of 10–15 molecules per reaction center. BChl 663 absorbs at 670 nm in vivo, with a specific extinction coefficient of 85 (±10) mM−1 · cm−1. In view of the evidence that the primary electron acceptor in P. aestuarii is a pigment with absorption maximum at 670 nm (Nuijs, A.M., Vasmel, H., Joppe, H.L.P., Duysens, L.N.M. and Amesz, J. (1985) Biochim. Biophys. Acta 807, 24–34) a direct consequence of these experiments is the fact that only BChl 663 can be a likely candidate for the role of primary electron acceptor as no other pigments absorbing around 670 nm (e.g., bacteriopheophytin c) are present in a photochemically active pigment-protein complex derived from the membrane of this green bacterium.

Optical properties of the photosynthetic reaction center of Chloroflexus aurantiacus at low temperature

Abstract A study was made of the optical properties of isolated reaction centers of the gliding green bacterium Chloroflexus aurantiacus. The absorption spectrum measured at 4 K indicates that these reaction centers contain bacteriochlorophyll a and bacteriopheophytin a, but no other pigments. The fluorescence excitation spectrum at 4 K showed efficient energy transfer from all pigment molecules to P-865, the primary electron donor. Absorption, linear and circular dichroism spectra of reduced and oxidized samples were measured at 77 K in order to obtain information about the dipole strengths, orientations and interactions of the transition dipoles. The results agree with a model for the reaction center that involves three bacteriopheophytin and four closely interacting bacteriochlorophyll molecules. The three bacteriopheophytins do not seem to be closely coupled to the primary donor. The primary electron donor consists of a bacteriochlorophyll dimer absorbing at 887 nm at 77 K, while the other two bacteriochlorophylls are responsible for the absorption around 800 nm and are also strongly coupled as indicated by their exciton splitting.

The triplet state of the primary donor of the green photosynthetic bacterium Chloroflexus aurantiacus

The technique of absorbance‐detected electron spin resonance in zero magnetic field (ADMR) was applied to investigate the structure of the reaction center of the facultatively aerobic green bacterium Chloroflexus aurantiacus. The triplet‐minus‐singlet absorbance difference spectrum thus obtained at 1.2K shows a clear resemblance to those earlier reported for Rhodopseudomonas viridis and Rps. sphaeroides R‐26. The most prominent features are the bleaching of the Qy band of the primary electron donor at 887 nm and the appearance of a narrow band at 807 nm upon triplet formation. We conclude that the primary electron donor P‐865 of Chloroflexus aurantiacus is a BChl a dimer with Qy and Qx absorbance bands at 887 and 606 nm, respectively, at 1.2 K; apparently the triplet state is localized on an optical time scale on one of the constituent pigments of the dimer. The zero field splitting parameters |D| and, |E| of P‐865 are 197.7 (± 0.7) × 10−4 cm−1 and 47.3 (± 0.7) × 10−4 cm−1, respectively. Decay rates of 12 660 (± 750) s−1, 14 290 (± 800) s−1 and 1690 (± 50) s−1 were observed for the x, y and z triplet sublevels, respectively.

Picosecond spectroscopy of the charge separation in reaction centers of Chloroflexus aurantiacus with selective excitation of the primary electron donor

Abstract Absorbance changes in the picosecond region were studied in isolated reaction centers of the green photosynthetic bacterium Chloroflexus aurantiacus upon selective excitation of the primary electron donor, P, at 870 nm. The results indicate that the first observed state is an excited state of P (P∗) which appears to transfer an electron to a bacteriochlorophyll a molecule absorbing at 812 nm (B 1 ) in 10 ± 2 ps as indicated by a bleaching at this wavelength. This reaction is followed by a rapid electron transfer (3 ± 1 ps) from B 1 − to bacteriopheophytin a , so that the fraction of reaction centers in the state P + B 1 − remains small during the experiment. An apparent bleaching at 925 nm is ascribed to stimulated emission from excited P, which emission disappears upon formation of P + . The difference between these time constants for electron transfer and those observed for the same reactions in reaction centers of the purple photosynthetic bacterium Rhodopseudomonas (Rhodobacter) sphaeroides is discussed in terms of the energy difference between P∗ and P + B 1 − , which appears to be larger for C. aurantiacus .

Triplet-minus-singlet absorbance difference spectra of reaction centers and antenna pigments of the green photosynthetic bacterium Prosthecochloris aestuarii

Abstract We have applied absorbance-detected electron spin resonance in zero magnetic field to several pigment-protein complexes that belong to the membrane-bound photosystem of the green sulfur bacterium Prosthecochloris aestuarii. It was found that three triplet states can be discerned, that are formed in the light-harvesting bacteriochlorophyll a protein, the core complex and in the primary donor P-840, respectively. Triplet-minus-singlet absorbance difference spectra of the latter two states are presented. The spectrum of the core complex shows a bleaching at 837 nm and an absorbance increase at 808 nm. This suggests a strong electronic interaction between at least two of the constituent BChl a molecules of the complex. The triplet-minus-singlet spectrum of P-840 shows two negative bands at 826 and 837 nm, that, according to their linear dichroism, have almost parallel polarization. It is shown that no spectral evidence exists for the presence of two resolved dimer exciton bands of P-840. We conclude that P-840 either consists of two weakly coupled BChl a molecules or of a strongly coupled pair with one allowed exciton band at 837 nm, the other blue-shifted exciton component being very weak. Decay rates of PT-840 of 6790 (±500) s−1, 3920 (±300) s−1 and 1275 (±100) s−1 were observed for the x, y and z triplet sublevels, respectively.