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

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.     

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.     

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

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.     

The primary charge separation,cytochrome oxidation and triplet formation in preparations from the green photosynthetic bacterium Prosthecochloris aestuarii

Flash-induced absorbance changes were measured in intact cells and subcellular preparations of the green photosynthetic bacterium Prosthecochloris aestuarii. In Complex I, a membrane vesicle preparation, photooxidation of the primary electron donor, P-840, and of cytochrome c-553 was observed. Flash excitation of the photosystem pigment complex caused in addition the generation of a bacteriochlorophyll a triplet. Triplet formation was the only reaction observed after flash excitation in the reaction center pigment -protein complex. The triplet had a lifetime of 90 μs at 295 K and of 165 μs at 120 K. The amount of triplet formed in a flash increased upon cooling from 295 to 120 K from 0.2 and 0.5 per reaction center to 0.45 and nearly 1 per reaction center in the photosystem pigment and reaction center pigment-protein complex, respectively. Measurements of absorbance changes in the near infrared in the reaction center pigment-protein complex indicate that the triplet is formed in the reaction center and that the reaction center bacteriochlorophyll a triplet is that of P-840. Formation of a carotenoid triplet did not occur in our preparations.Illumination with continuous light at 295 K of the reaction center pigment-protein complex produced a stable charge separation (with oxidation of P-840 and cytochrome c-553) in each reaction center, but with a low efficiency. This low efficiency, and the high yield of triplet formation is probably due to damage of the electron transport chain at the acceptor side of the reaction center of the reaction center pigment-protein complex.The halftime for cytochrome c-553 oxidation in Complex I and the photosystem pigment complex was 90 μs at 295 K; below 220 K no cytochrome oxidation occurred. At 120 K P-840⁺ was rereduced with a halftime of 20 ms, presumably by a back reaction with a reduced acceptor.     

Antenna organization and evidence for the function of a new antenna pigment species in the green photosynthetic bacterium Chloroflexus aurantiacus

Whole cells and isolated chlorosomes (antenna complex) of the green photosynthetic bacterium Chloroflexus aurantiacus have been studied by absorption spectroscopy (77 K and room temperature), fluorescence spectroscopy, circular dichroism, linear dichroism and electron spin resonance spectroscopy. The chlorosome absorption spectrum has maxima at 450 (contributed by carotenoids and bacteriochlorophyll (BChl) a Soret), 742 (BChl c) and 792 nm (BChl a) with intensity ratios of 20:25. The fluorescence emission spectrum has peaks at 748 and 802 nm when excitation is into either the 742 or 450 nm absorption bands, respectively. Whole cells have fluorescence peaks identical to those in chlorosomes with the addition of a major peak observed at 867 nm. The CD spectrum of isolated chlorosomes has an asymmetric-derivative-shaped CD centered at 739 nm suggestive of exciton interaction at least on the level of dimers. Linear dichroism of oriented chlorosomes shows preferential absorption at 742 nm of light polarized parallel to the long axis of the chlorosome. This implies that the transition dipoles are also oriented more or less parallel to the long axis of the chlorosome. Treatment with ferricyanide results in the appearance of a 2.3 G wide ESR spectrum at g 2.002. Whole cells grown under different light conditions exhibit different fluorescence behavior when absorption is normalized at 742 nm. Cells grown under low light conditions have higher fluorescence intensity at 748 nm and lower intensity at 802 nm than cells grown under high light conditions. These results indicate that the BChl c in chlorosomes is highly organized, and transfers energy from BChl c (742 nm) to a connector of baseplate BChl B792 (BChl a) presumably located in the chlorosome baseplate adjacent to the cytoplasmic membrane.     

Evidence for a new early acceptor in Photosystem I of plants. An ESR investigation of reaction center triplet yield and of the reduced intermediary acceptors

The yield of the triplet state of the primary electron donor of Photosystem I of photosynthesis (P^T-700) and the characteristic parameters (g value, line shape, saturation behavior) of the ESR signal of the photoaccumulated intermediary acceptor A have been measured for two types of Photosystem I subchloroplast particles: Triton particles (TSF 1, about 100 chlorophyll molecules per P-700) that contain the iron-sulfur acceptors F_X, F_B and F_A, and lithium dodecyl sulfate (LDS) particles (about 40 chlorophyll molecules per P-700) that lack these iron-sulfur acceptors. The results are: (i) In Triton particles the yield of P^T-700 upon illumination is independent of the redox state of A and of F_X,B,A and is maximally about 5% of the active reaction centers at 5 K. The molecular sublevel decay rates are k_x = 1100 s^?1 ± 10%, k_y = 1300 s^?1 ± 10% and k_z = 83 s^?1 ± 20%. In LDS particles the triplet yield decreases linearly with concentration of reduced intermediary acceptors, the maximal yield being about 4% at 5 K assuming full P-700 activity. (ii) In Triton particles the acceptor complex A consists of two acceptors A₀ and A₁, with A₀ preceding A₁. In LDS particles at temperatures below ?30°C only A₀ is photoactive. (iii) The spin-polarized ESR signal found in the time-resolved ESR experiments with Triton particles is attributed to a polarized P-700-A^?₁ spectrum. The decay kinetics are complex and are influenced by transient nutation effects, even at low microwave power. It is concluded that the lifetime at 5 K of P-700A₀A^?₁ must exceed 5 ms. We conclude that P^T-700 originates from charge recombination of P-700A^?₀, and that in Triton particles A₀ and A₁ are both photoaccumulated upon cooling at low redox potential in the light. Since the state P-700AF^?_X does not give rise to triplet formation the 5% triplet yield in Triton particles is probably due to centers with damaged electron transport.     

Singlet and triplet excited carotenoid and antenna bacteriochlorophyll of the photosynthetic purple bacterium Rhodospirillum rubrum as studied by picosecond absorbance difference spectroscopy

Picosecond absorbance difference spectra at a number of delay times after a 35 ps excitation pulse and kinetics of absorbance changes were measured in chromatophores of the photosynthetic purple bacterium Rhodospirillum rubrum after chemical oxidation of the primary electron donor P-875. Kinetics and spectra were measured of the excited singlet states of carotenoid and bacteriochlorophyll a and also of the triplet state of the carotenoid. The excited singlet state of carotenoid, produced by direct excitation at 532 nm, is characterized by a bleaching of the ground state absorption bands in the region 450–490 nm and by an absorbance increase with a maximum near 570 nm. Its lifetime was calculated to be 0.6 ± 0.1 ps in vitro and less than 1 ps in vivo. The triplet state of carotenoid in vivo is formed within 100 ps after direct carotenoid excitation via a pathway that does not involve excited states of bacteriochlorophyll. Singlet excitation of a bacteriochlorophyll a molecule causes the bleaching of its Q_x and Q_y absorption bands, and is probably associated with blue shifts of the Q_y absorption band of about six neighboring bacteriochlorophyll molecules. Upon increasing the excitation density, the average lifetime of the singlet excitations on bacteriochlorophyll decreased from about 350 ps to about 10 ps or less. The results are in quantitative agreement with the known effect of singlet-singlet annihilation upon the fluorescence yield, and furthermore show that no bacteriochlorophyll or carotenoid triplet formation is associated with this annihilation.     

Triplet-minus-singlet absorbance difference spectra of reaction centers of Rhodopseudomonas sphaeroides R-26 in the temperature range 24–290 K measured by Magneto-Optical Difference Spectroscopy (MODS)

The recently developed technique of Magneto-Optical Difference Spectroscopy (MODS) [10] has been applied to reaction centers (RC) of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26. Absorbance changes induced by a magnetic field are measured as a function of wavelength yielding the triplet-minus-singlet (T-S) absorbance difference spectrum. (T-S) spectra thus obtained have been measured from 24–290 K. Going from low to high temperature the (T-S) spectra show the following features:

1. A rapid decrease of positive absorption bands at 809 and 819 nm.
2. A slow appearance of a band shift at 798 nm.
3. A shift of the peak wavelength of the Q_y absorbance band of the primary donor P-860 from 992 to 861 nm, and of its Q_x band from 603 to 600 nm.

The spectra at 24, 66, 116, and 290 K have been analyzed by Gaussian deconvolution. The 800 nm region of the spectrum at 24 K can be decomposed in a combination of two band shifts and an appearing band. The temperature dependence of the spectra in this region is well explained by spectral broadening of the two shifting bands combined with a decrease in intensity of the appearing band when the temperature increases. The two shifting bands in the 800 nm region are identified as the two bands at 803 and 813 nm which together make up the 800 nm band in the absorption spectrum and are assigned to the two accessory RC bacteriochlorophylls (BChls). The band shift of the 813 nm pigment is appreciably larger than that of the 803 nm pigment. The appearing band at 808 nm is attributed to monomeric absorption of ³P-860, the triplet state being localized on one BChl. We find no evidence for admixture of a charge transfer (CT) state of ³P-860 with one of the accessory BChls at higher temperature.     

Linear-dichroic triplet-minus-singlet absorbance difference spectra of reaction centers of the photosynthetic bacteria Chromatium vinosum,Rhodopseudomonas sphaeroides R-26 and Rhodospirillum rubrum S1

For the first time, linear-dichroic triplet-minus-singlet (LD-(T - S)) spectra of reaction centers of the photosynthetic bacteria Chromatium vinosum, Rhodopseudomonas sphaeroides R-26 and Rhodospirillum rubrum S1 have been measured using an extension of the technique of absorbance-detected magnetic resonance (ADMR) of the triplet state. For all bacteria studied the LD-(T - S) spectra exhibit a bleaching of the long-wavelength absorbance band that is either split or has a clear shoulder to longer wavelengths. The components are approximately parallel-polarized, indicating that they do not form an exciton pair. Around 800 nm a band appears with a width of about 7 nm, which does not form part of a band shift and that may be attributed to an appearing monomer band. Small features in the LD-(T - S) spectra at both sides of this band are well explained by band shifts of the two components of the 800 nm reaction center absorption band. The transition moment of the component at about 818 nm in reaction centers of Rps. sphaeroides R-26 is at an angle larger than 55° with both the x and the y triplet spin axes. In none of the bacteria do we find evidence for the bleaching of an exciton component of P-860 near 810 nm.     

The primary electron acceptor of green sulfur bacteria,bacteriochlorophyll 663, is chlorophyll a esterified with Δ 2,6-phytadienol

The primary electron acceptor of green sulfur bacteria, bacteriochlorophyll (BChl) 663, was isolated at high purity by an improved purification procedure from a crude reaction center complex, and the molecular structure was determined by fast atom bombardment mass spectroscopy (FAB-mass), ¹H- and ¹³C-NMR spectrometry, double quantum filtered correlation spectroscopy (DQF-COSY), heteronuclear multiple-quantum coherence (HMQC) and heteronuclear multiple-bond correlation (HMBC) spectral measurements. BChl 663 was 2.0 mass units smaller than plant Chl a. The NMR spectra showed that the macrocycle was identical to that of Chl a. In the esterifying alcohol, a singlet P7¹ signal was observed at the high-field side of the singlet P3¹ signal in BChl 663, while a doublet peak of P7¹ overlapped that of P11¹ in Chl a. A signal of P7-proton, seen in Chl a, was lacking, and the P6-proton appeared as a triplet signal near the triplet P2-proton signal in BChl 663. These results indicate the presence in BChl 663 of a C=C double bond between P6 and P7 in addition to that between P2 and P3. The structure of BChl 663 was hence concluded to be Chl a esterified with 2,6-phytadienol instead of phytol. In addition to BChl 663, two molecules of the 13²-epimer of BChl a, BChl a′, were found to be present per reaction center, which may constitute the primary electron donor. This revised version was published online in June 2006 with corrections to the Cover Date.     

Utilization of blue-green light by chlorosomes from the photosynthetic bacterium Chloroflexus aurantiacus: Ultrafast excitation energy conversion and transfer

Chlorosomes of photosynthetic green bacteria are unique molecular assemblies providing efficient light harvesting followed by multi-step transfer of excitation energy to reaction centers. In each chlorosome, 10⁴–10⁵ bacteriochlorophyll (BChl) c/d/e molecules are organized by self-assembly into high-ordered aggregates. We studied the early-time dynamics of the excitation energy flow and energy conversion in chlorosomes isolated from Chloroflexus (Cfx.) aurantiacus bacteria by pump-probe spectroscopy with 30-fs temporal resolution at room temperature. Both the S₂ state of carotenoids (Cars) and the Soret states of BChl c were excited at ~490 nm, and absorption changes were probed at 400–900 nm. A global analysis of spectroscopy data revealed that the excitation energy transfer (EET) from Cars to BChl c aggregates occurred within ~100 fs, and the Soret → Q energy conversion in BChl c occurred faster within ~40 fs. This conclusion was confirmed by a detailed comparison of the early exciton dynamics in chlorosomes with different content of Cars. These processes are accompanied by excitonic and vibrational relaxation within 100–270 fs. The well-known EET from BChl c to the baseplate BChl a proceeded on a ps time-scale. We showed that the S₁ state of Cars does not participate in EET. We discussed the possible presence (or absence) of an intermediate state that might mediates the Soret → Q_y internal conversion in chlorosomal BChl c. We discussed a possible relationship between the observed exciton dynamics and the structural heterogeneity of chlorosomes.     

Excitation transfer in chlorosomes of green photosynthetic bacteria

The formation of excited states and energy transfer in chlorosomes of the green photosynthetic bacteria Chlorobium limicola and Chloroflexus aurantiacus were studied by measurements of flash-induced absorbance changes and fluorescence. Upon excitation with 35 ps, 532 nm flashes, large absorbance decreases around 750 nm were observed that were due to the disappearance of ground state absorption of the main pigment, bacteriochlorophyll (BChl) c. The absorbance changes decayed after the flash with a time constant of approx. 1 ns, together with faster components. Absorbance changes that could be ascribed to formation of excited BChl a were much smaller than those of BChl c. The yields of BChl c and BChl a fluorescence were measured as a function of the energy density of the exciting flash. At high energy a strong quenching occurred caused by annihilation of singlet excited states. An analysis of the results shows that energy transfer between BChl c molecules is very efficient and that in C. limicola excitations can probably move freely through the entire chlorosome (which contains about 10 000 BChls c). The chlorosome thus serves as a common antenna for several reaction centres. The small amounts of BChl a present in the chlorosomes of both species form clusters of only a few molecules. Upon cooling to 4 K the sizes of the domains of BChl c for energy transfer decreased considerably. The results are discussed in relation to recently suggested models for the pigment organization within chlorosomes.     

Excitation Energy Transfer Dynamics and Excited-State Structure in Chlorosomes of Chlorobium phaeobacteroides

The excited-state relaxation within bacteriochlorophyll (BChl) e and a in chlorosomes of Chlorobium phaeobacteroides has been studied by femtosecond transient absorption spectroscopy at room temperature. Singlet-singlet annihilation was observed to strongly influence both the isotropic and anisotropic decays. Pump intensities in the order of 10¹¹ photons × pulse⁻¹ × cm⁻² were required to obtain annihilation-free conditions. The most important consequence of applied very low excitation doses is an observation of a subpicosecond process within the BChl e manifold (~200–500 fs), manifesting itself as a rise in the red part of the Q_y absorption band of the BChl e aggregates. The subsequent decay of the kinetics measured in the BChl e region and the corresponding rise in the baseplate BChl a is not single-exponential, and at least two components are necessary to fit the data, corresponding to several BChl e→BChl a transfer steps. Under annihilation-free conditions, the anisotropic kinetics show a generally slow decay within the BChl e band (10–20 ps) whereas it decays more rapidly in the BChl a region (~1 ps). Analysis of the experimental data gives a detailed picture of the overall time evolution of the energy relaxation and energy transfer processes within the chlorosome. The results are interpreted within an exciton model based on the proposed structure.     

The photochemical reaction center of the bacteriochlorophyll b-containing organism Thiocapsa pfennigii

A photochemical reaction-center preparation has been made from a second bacteriochlorophyll b-containing organism, Thiocapsa pfennigii. The reaction-center unit is thought to be composed of one P-960, four bacteriochlorophyll, two bacteriopheophytin, one carotenoid molecules and polypeptides of M_r 40000, 37000, 34000, 27000 and 26000 probably plus quinones and metal atoms. The preparation also contains a low-potential cytochrome c-555 and a high-potential cytochrome c-557 bound to the reaction center in a 3–4:2–3:1 molar ratio with respect to P-960. The 40 kDa subunit is associated with the cytochromes, while the 37, 34 and 27 + 26 kDa subunits are proposed to be equivalent to the H, M and L polypeptides of bacteriochlorophyll a-containing reaction centers. The cytochromes are oxidized by P-960⁺. The three near-infrared absorption bands at 788, 840 and 968 nm are assigned to bacteriopheophytin, bacteriochlorophyll and the primary donor (P-960), respectively. The 778 nm peak resolves into two at 77 K; no further resolution of the other two peaks occurs. Illumination of the sodium dithionite-reduced reaction centers at 77 K by 960 nm-light results in P-960, transferring one electron from cytochrome c-555 mainly to a bacteriopheophytin molecule, absorbing at 781 nm. A similar treatment at room temperatures reduces most of the two bacteriopheophytin molecules. It is argued that both bacteriopheophytin molecules, possibly with some contribution from bacteriochlorophyll, form an intermediary electron-carrier complex between P-960 and a quinone in T. pfennigii. We could not substantiate that a bacteriochlorophyll molecule precedes the bacteriopheophytins in the electron transfer sequence. Although the biochemical characteristics of the reaction center are very similar to those of the other known bacterioclorophyll b-containing reaction center, that from Rhodopseudomonas viridis, their spectral characteristics are not. This has helped elucidate more about the function of each spectral form and led us to conclude that the 850 nm form in Rps. viridis is not the higher energy transition of the special pair of bacteriochlorophyll molecules forming P-960. Laser-flash-in-duced absorbance changes in T. pfennigii reaction-center preparation should now lead to a more complete understanding of the mechanism of the primary photochemical event.     

Absorption and linear dichroism spectroscopy at room and low temperatures of single crystals of the B800–850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050

The absorbance, polarized absorbance and linear dichroism spectra of single crystals of the B800–850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050 taken at room (298 K) and low (85 K) temperatures are presented. The spectra are compared and contrasted with random phase solution spectra from the same complex. The single crystal spectra display a spectral narrowing at low temperatures in the BChl Q_x (550–650 nm) and carotenoid (450–550 nm) regions similar to that observed from the random phase solution. The single crystal absorption spectra in the BChl Q_y (750–900 nm) region are broader than the solution spectra and remain broad as the temperature is lowered. It is suggested that this broadening is the result of specific exciton interactions between the BChl chromophore Q_y transition dipoles and is a molecular feature which occurs only in the crystalline complex.     

Chemical oxidation of the FMO antenna protein from Chlorobaculum tepidum

Effect of chemical oxidation by ferricyanide on bacteriochlorophyll a (BChl a) in the Fenna–Matthews–Olson protein (FMO) was studied using absorbance and fluorescence spectroscopy at ambient and cryogenic temperatures. Partially selective oxidation of pigments bound to the antenna complex was achieved and the probable absorption wavelength corresponding to the recently discovered bacteriochlorophyll No. 8 of 806 nm was obtained by comparative analysis of the effect of chemical oxidation and the effect of different isolation procedures. Formation of a stable product identified as a chlorophyll a derivative occurred upon chemical oxidation. This new pigment remained bound within the pigment–protein complex, and exhibited an efficient energy transfer to BChl a. Furthermore, complex effects of the pigment oxidation upon the fluorescence yield of the FMO protein were observed. Utility of this approach based on chemical modifications for the investigation of the native regulatory mechanisms involved in the energy transfer in the FMO protein is discussed.     

Kinetics of picosecond bacteriochlorophyll luminescence in vivo as a function of the reaction center state

Bacteriochlorophyll (BChl) luminescence lifetimes (τ) were measured in purple bacteria Rhodospirillum rubrum and Rhodopseudomonas sphaeroides at low-excitation pulse energy with the use of a picosecond luminescence spectrochronograph of high sensitivity and high time-resolution. Average high-frequency excitation light density was changed from about 1 · 10¹³ photons · cm⁻² · s⁻¹ up to 1 · 10¹⁷ photons · cm⁻². s⁻¹. Maximal energy density in a single pulse was in the range 10⁻¹⁴–10⁻¹⁰ J/cm², which completely rules out nonlinear exciton interactions. In this range τ increased as a function of excitation light density from about 60 ps to 210 ps. Luminescence yield (ø) for the bacteria investigated measured under continuous or picosecond excitation changed in a similar manner as τ. The luminescence increase was shown to accompany the conversion of the reaction centers to the closed, photooxidized state. Luminescence decay of R. rubrum and Rps. sphaeroides chromatophores without any chemical additions was well approximated by a single exponential component both at low and at saturating intensities of exciting light. The time necessary for the primary charge separation to occur was shown to be 60 ± 10 ps. The pairwise jump-time of excitation-energy transfer, as well as excitation-diffusion characteristics were estimated from these data. On the basis of life-time measurements in the state of active photosynthesis, the quantum yield of the primary charge separation in the reaction centers was estimated to be equal to 0.95 ± 0.02. In intact cells as well as in chromatophores in the presence of reducing agents, a nanosecond component of emission decay was also observed. The relative amplitude of this component, being several percent of the picosecond one at low-excitation intensity levels, increased (2–3)-times with excitation density. Its life-time was estimated to be 3 ± 1 ns. The nanosecond component appeared only under conditions when a part of the reaction centers were converted to the closed state PQ⁻.     

Interactions of steroids with human placental cytochrome P-450 in the presence of carbon monoxide.

Spectral analyses of the carbon monoxide (CO) complex of human placental microsomal cytochrome P-450 revealed absorption maxima at 426 and 450 nm when NADPH (2×10⁻⁴M) was utilized as a reducing agent. Additional NADPH or NADH did not produce any further increases in the absorption maximum at 450 nm. A period of 10–15 minutes was required for the complete reduction. Various steroids were added to both sample and reference cuvettes to examine their interactions with the CO-cytochrome P-450 complex. The resulting spectral changes indicated that low concentrations of steroids (≃10⁻⁷M) such as androstenedione, 19-hydroxyandrostenedione, 19-oxoandrostenedione and testosterone completely eliminated the absorbance maxima at 450 nm while 19-norandrostenedione, 19-nortestosterone, pregnenolone and benzo[a]-pyrene did not eliminate this peak. Since ample time was allowed to reduce the cytochrome P-450 with NADPH, the observed interaction of steroids with cytochrome P-450 in the presence of CO does not represent an effect on reductase activity, but on the formation of the CO-cytochrome P-450 complex.     

Spectral and photochemical properties of borohydride-treated D1-D2-cytochrome b-559 complex of photosystem II

The D1-D2-cytochrome b-559 reaction center complex of photosystem II with an altered pigment composition was prepared from the original complex by treatment with sodium borohydride (BH⁻₄). The absorption spectra of the modified and original complexes were compared to each other and to the spectra of purified chlorophyll a and pheophytin a (Pheo a) treated with BH⁻₄ in methanolic solution. The results of these comparisons are consistent with the presence in the modified complex of an irreversibly reduced Pheo a molecule, most likely 13¹-deoxo-13¹-hydroxy-Pheo a, replacing one of the two native Pheo a molecules present in the original complex. Similar to the original preparation, the modified complex was capable of a steady-state photoaccumulation of Pheo⁻ and P680⁺. It is concluded that the pheophytin a molecule which undergoes borohydride reduction is not involved in the primary charge separation and seems to represent a previously postulated photochemically inactive Pheo a molecule. The Q_y and Q_x transitions of this molecule were determined to be located at 5°C at 679.5–680 nm and 542 nm, respectively.     

Initial characterization of the photosynthetic apparatus of "Candidatus Chlorothrix halophila," a filamentous, anoxygenic photoautotroph

“Candidatus Chlorothrix halophila” is a recently described halophilic, filamentous, anoxygenic photoautotroph (J. A. Klappenbach and B. K. Pierson, Arch. Microbiol. 181:17-25, 2004) that was enriched from the hypersaline microbial mats at Guerrero Negro, Mexico. Analysis of the photosynthetic apparatus by negative staining, spectroscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the photosynthetic apparatus in this organism has similarities to the photosynthetic apparatus in both the Chloroflexi and Chlorobi phyla of green photosynthetic bacteria. The chlorosomes were found to be ellipsoidal and of various sizes, characteristics that are comparable to characteristics of chlorosomes in other species of green photosynthetic bacteria. The absorption spectrum of whole cells was dominated by the chlorosome bacteriochlorophyll c (BChl c) peak at 759 nm, with fluorescence emission at 760 nm. A second fluorescence emission band was observed at 870 nm and was tentatively attributed to a membrane-bound antenna complex. Fluorescence emission spectra obtained at 77 K revealed another complex that fluoresced at 820 nm, which probably resulted from the chlorosome baseplate complex. All of these results suggest that BChl c is present in the chlorosomes of “Ca. Chlorothrix halophila,” that BChl a is present in the baseplate, and that there is a membrane-bound antenna complex. Analysis of the proteins in the chlorosomes revealed an ~6-kDa band, which was found to be related to the BChl c binding protein CsmA found in other green bacteria. Overall, the absorbance and fluorescence spectra of “Ca. Chlorothrix halophila” revealed an interesting mixture of photosynthetic characteristics that seemed to have properties similar to properties of both phyla of green bacteria when they were compared to the photosynthetic characteristics of Chlorobium tepidum and Chloroflexus aurantiacus.