The role of the accessory bacteriochlorophyll in reaction centers of photosynthetic bacteria: intermediate acceptor in the primary electron transfer?

Time-resolved spectroscopy in conjunction with magnetic-field-dependent recombination dynamics of the primary radical ion pair in reaction centers of Rb, sphaeroides R26, were used to analyze the mechanism of electron transfer from the bacteriochlorophyll dimer in its excited singlet state (¹P^*) to bacteriopheophytin (H). This analysis provides evidence against the participation of the accessory bacteriochlorophyll (B) as a kinetic intermediate and thus favours a single-step electron transfer, which is mediated by superexchange electronic interactions.     

Excited-state absorption and ultrafast relaxation dynamics of porphyrin, diprotonated porphyrin, and tetraoxaporphyrin dication

The relaxation dynamics of unsubstituted porphyrin (H2P), diprotonated porphyrin (H4P2+), and tetraoxaporphyrin dication (TOxP2+) has been investigated in the femtosecond-nanosecond time domain upon photoexcitation in the Soret band with pulses of femtosecond duration. By probing with spectrally broad femtosecond pulses, we have observed transient absorption spectra at delay times up to 1.5 ns. The kinetic profiles corresponding with the band maxima due to excited-state absorption have been determined for the three species. Four components of the relaxation process are distinguished for H2P: the unresolvably short B --> Qy internal conversion is followed by the Qy --> Qx process, vibrational relaxation, and thermalization in the Qx state with time constant approximately 150 fs, 1.8 ps, and 24.9 ps, respectively. Going from H2P to TOxP2+, two processes are resolved, i.e., B --> Q internal conversion and thermal equilibration in the Q state. The B --> Q time constant has been determined to be 25 ps. The large difference with respect to the B --> Qy time constant of H2P has been related to the increased energy gap between the coupled states, 9370 cm-1 in TOxP2+ vs 6100 cm-1 in H2P. The relaxation dynamics of H4P2+ has a first ultrafast component of approximately 300 fs assigned as internal conversion between the B (or Soret) state and charge-transfer (CT) states of the H4P2+ complex with two trifluoroacetate counterions. This process is followed by internal CT --> Q conversion (time constant 9 ps) and thermalization in the Q state (time constant 22 ps).     

Spectroscopic studies on self-aggregation of bacteriochlorophyll-e in nonpolar organic solvents: effects of stereoisomeric configuration at the 3(1)-position and alkyl substituents at the 8(1)-position.

Self-aggregation of naturally occurring bacteriochlorophyll (BChl)-e in nonpolar organic solvents was investigated by visible absorption, fluorescence emission and circular dichroism spectra. Cultured brown-colored photosynthetic bacteria have several BChl-e as light-harvesting antenna pigments. Three major BChl-e homologs were separated from the extracts of the culture by reverse-phase high-performance liquid chromatography (HPLC) and characterized by 1H-NMR and fast-atom bombardment mass spectroscopy: 8-ethyl-12-ethyl ([E,E])-, 8-propyl-12-ethyl- and 8-isobutyl-12-ethyl-BChl-e farnesyl esters. All the homologs consisted of a mixture of the 3(1)-epimers, and epimerically pure BChl-e were also given by HPLC separation. All the separated BChl-e epimers, the epimeric mixtures and the homologous mixtures formed self-aggregates in 2% dichloromethane/hexane, giving visible absorption spectra similar to that of the whole cells, which showed two peaks (or shoulders) around 430-450 and 520 nm at the Soret region as well as a red-shifted Qy band relative to the monomeric. The spectral properties of the Soret band were basically unchanged among the epimers or epimeric/homologous mixtures. In contrast, the Qy band of aggregates of epimeric mixtures (except [E,E]) and homologous mixtures red-shifted and broadened compared with the epimerically pure. The red-shift and broadening of the Qy band are advantageous for efficient energy transfer from BChl-e aggregates to BChl-a in a baseplate in chlorosomes because their spectral overlap increases.     

Planarity of metal chlorins in n-octane solution

The geometry of porphyrins has been and continues to be a subject of vigorous research. In this work we investigated the planarity of a series of divalent metal chlorins in n-octane solutions: magnesium, zinc, copper, nickel, cobalt, manganese, tin, cadmium, strontium, lead and platinum. Room temperature electronic absorption spectra of these complexes are reported. We plotted the energy of each B band against its respective Qy origin band. Based on these results it was concluded that in these metal chlorins the configuration interaction between Qy and B states is essentially constant; the exception is the magnesium complex which deviates from the pattern produced by the other metal chlorins. This suggests that the magnesium ion is out of the plane of the chlorin skeleton; thus, it has C(S) symmetry while the other metal chlorins in this group retained C2v symmetry.     

NEAR-INFRARED-FT-RAMAN STUDY OF AGGREGATION OF BACTERIOCHLOROPHYLL c IN WHOLE LIVING Chlorobium limicola

Near-infrared excited Fourier-transform Raman spectra have been measured for whole living Chlorobium limicola f. thiosulfatophilum to explore in situ structure of bacteriochlorophyll c (BChl-c). The spectra, whose Raman bands are preresonance enhanced via a Qy band of BChl-c, did not contain contributions from the major components of bacteria such as proteins and lipids. Therefore, the spectra provide selective structural information about BChl-c in the chlorosomes in a totally nondestructive manner. A marker band, appearing at 1605 cm-1 for the coordination number of the Mg atom, shows that BChl-c in the chlorosomes is five coordinate. The Raman spectrum of living bacteria closely resembles that of BChl-c in water-saturated carbon tetrachloride (w-std CC14) where it is comprised of dimer, tetramer and polymer spectra. However, a band assigned to a C=O stretching mode of the 131-keto group is identified only at 1641 cm-1. A band arising from the free keto carbonyl group, which appears in the spectrum of BChl-c in w-std CC14, is not observed in the spectra of bacteria. These observations suggest that BChl-c in the chlorosomes forms mostly coordinate polymeric species whose structure is very similar to that of BChl-c in w-std CC14.     

Synthesis and optical properties of bacteriochlorophyll-a derivatives having various C3 substituents on the bacteriochlorin pi-system

Methyl bacteriopyropheophorbide-a derivatives having a series of substituents at the C3 position were prepared and their optical properties were compared with the corresponding chlorin analogues. Two kinds of oxidation reaction (C3-vinyl --> formyl --> carboxy group) were found to be applicable with a little alteration of the free-base bacteriochlorin macrocycles. The Qx and Qy electronic absorption peak positions of synthetic bacteriochlorins in CH2Cl2 were affected by the C3 substituents and found to be more sensitive than those of the chlorins. The observed Qx/Qy peaks in their monomeric states were shifted to a longer wavelength in the order of 1-hydroxyethyl < hydroxymethyl < acetoxymethyl < vinyl < acetyl < carboxy < formyl < 2,2-dicyanoethynyl group. Zinc complex with the C3-hydroxymethyl group formed self-aggregates in a nonpolar organic solvent, which showed the largest red-shift of the Qy band (2380 cm(-1), 726 nm in THF to 878 nm in 1% THF-cyclohexane) among those of the synthetic self-aggregative (bacterio)chlorins examined.     

Bakteriochlorophyll aGg und Bakteriophäophytin aP in den photosynthetischen Reaktionszentren von rhodospirillum rubrum G-9+

Bacteriochlorophyll a_Gg and Bacteriopheophytin a_p in Photosynthetic Reaction Centers from Rhodospirillum rubrum G-9⁺ In photosynthetically active reaction centers from Rhodospirillum rubrum G-9⁺, the magnesium complex bacteriochlorophyll a contains geranylgeraniol as the alcohol component, while the metal-free bacteriopheophytin a contains phytol instead. These pigments bacteriochlorophyll a_Gg ( 4 ) and bacteriopheophytin a_P ( 1 ) were isolated from reaction center preparations in a ratio of 2:1 and (after demetallation of 4 ) identified as bacteriopheophytin a_Gg ( 2 ), and a_P ( 1 ) by comparison with authentic samples (UV./VIS., CD. and mass spectra as well as mixed HPLC.).     

HL-LH2中色素分子间的单重激发态能量传递

采用飞秒时间分辨吸收光谱手段观测了在500和800 nm激发下高光培养的紫色光合细菌Rhodopseu-domonas(Rps). palustris外周捕光天线LH2(HL-LH2)中不同共轭链长类胡萝卜素(Carotenoid, 简称Car)和细菌叶绿素a(Bacteriachlorophyll a, 简称BChl a)的特征吸收光谱. 光谱动力学分析结果表明, HL-LH2中不同Car分子间可能存在复杂的单重激发态能量平衡过程, Car分子同时向BChl a分子发生多途径的单重激发态能量传递, B800主要接受来自Car的S2和S1态能量; B850则主要接受来自长共轭链Car(共轭双键数目n=13)的S1态和B800的激发态能量, 整个能量传递过程在3~5 ps内完成.     

Effect of denaturants on the stability of light-harvesting complex

 The effect of denaturants such as urea and normal alcohols on the formation of light-harvesting (LH) polypeptides/bacteriochlorophyll a (BChla) complex (LH1 complex) in n-octyl-β-D-glucopyranoside (OG) micelle was examined to provide an insight into stability of the complex. The stabilities of the LH1 complex in OG micelle and of the complex in the chromatophore of photosynthetic bacteria were compared by addition of denaturants. The extent of stability of these complexes was monitored by the change in absorbance of Qy band of BChla in these complexes, resulting generally in the blue-shifting of the Qy band from near 870 nm to about 777 nm upon addition of these denaturants. Urea and guanidium hydrochloride (Gnd) showed a relatively weak denaturing effect. Normal alcohols showed stronger denaturing effect, depending on the hydrophobicity of the alcohols. These results imply that the stability of LH1 complex in OG micelle can be largely attributed to the hydrophobic interactions in the complex as well as that of the complex in the chromatophore of photosynthetic bacteria. Received: 23 May 1997 Accepted: 13 September 1997     

Dipole strengths of the Qy(0,0) bacteriochlorophyll c transition

The absorption spectra of bacteriochlorophyll (BChl) c solutions in two mixtures of two solvents (acetonitrile with pyridine and dimethylsulfoxide with methanol) exhibiting different refractive indices were measured and deconvoluted into Gaussian components. The refractive index of mixed solvents was changed by the change in the ratio of the volumes of the liquids used in the mixture. Using the Qy(0,0) band half widths and absorption coefficient, based on the simplified formula proposed by Knox, the dipole strengths of the Qy(0,0) BChl c transition for various values of solvent refractive index were calculated and compared with values given by Knox and Spring. For both investigated combinations of two liquids, the dependence of Qy(0,0) transition dipole strength of the BChl c on solvent refractive index was almost linear. The slopes of the lines obtained from the experimental absorption bands were different for two investigated solvent mixtures. More accurate linear dependence and similar slopes of lines for both solvent mixtures were obtained using half widths and absorption coefficients of the Gaussian components of Qy(0,0) transition. It is explained by the superposition of the contributions from other electronic and vibronic transitions of BChl c monomers or possibly also from transitions of the pigments involved in some complexes with solvent molecules in the absorption region investigated. The results show that the formula proposed by Knox can be successfully applied also for BChl c, after elimination of the overlapped contributions from the other transitions, by applying Gaussian analysis to select only contribution from Qy(0,0) pigment transition.     

Vibrational spectroscopy and intramolecular dynamics of 1-butyne

Photodissociation of jet-cooled vibrationally excited 1-butyne, C(2)H(5)C[Triple Bond]C[Single Bond]H, coupled with mass spectrometric detection of H photofragments, facilitated measurements of action spectra and Doppler profiles, expressing the yield of the ensuing fragments versus the vibrational excitation and UV probe lasers, respectively. Both the action spectra and the simultaneously measured room temperature photoacoustic spectra in the 2nu(1), 3nu(1), and 4nu(1) C[Single Bond]H acetylenic stretch regions exhibit unresolved rotational envelopes with significant narrowing of the former due to temperature-related change in the rotational structure. The narrowing of the action spectrum in the 3nu(1) region exposed a resonance splitting, implying intramolecular vibrational energy redistribution (IVR) time of approximately 1 ps. Asymmetric rotor simulation of the band contours provided the rotational constants and estimates for the homogeneous broadening arising from IVR to the bath vibrational states. The homogenous linewidth of 4nu(1) is anomalously narrower than that of 2nu(1) and 3nu(1), indicating a longer lived 4nu(1) state despite the increasing background state density, suggestive of a lack of low-order resonances or of mode-specific coupling with the bath states. The Doppler profiles indicate that the H photofragments are released with low average translational energies, pointing to an indirect dissociation process occurring after internal conversion (IC) to the ground electronic state or after IC and isomerization to butadiene.     

ABSORPTION OF METHYLBACTERIOPHEOPHORBIDE a SINGLE CRYSTALS: SPECTRAL SHIFTS DUE TO π-π INTERACTIONS

Abstract— Polarized absorption spectra were obtained for a single crystal of methylbacteriophorbide a (MeBPhide a). The Q_y band is red-shifted ∽ 1660 cm^-1 (∽ 110 nm) relative to MeBPhide a in a CH₂Cl₂/benzene solution. This is equivalent to the largest red shifts observed for in vivo bacteriochlorophyll a. The Soret band exhibits a smaller red shift and a significant reduction in intensity, and the Q_x band is not observed. The crystal spectra are qualitatively similar to spectra reported for several other aggregated (bacterio)chlorophyll and bacteriopheophytin systems. Since crystalline MeBPhide a contains no Mg, water or hydrogen bonding (Barkigia etal. , 1981), these results demonstrate that the spectral changes associated with the aggregation of photosynthetic chromophores can arise solely from IT-IT interactions between macrocycles.     

Time-resolved spectroscopy of energy transfer and trapping upon selective excitation in membranes of Heliobacillus mobilis at low temperature

Transient absorption difference spectra in the Qy absorption band of bacteriochlorophyll (BChl) g and in the 670 nm absorption band of the primary acceptor A0 in membranes of Heliobacillus mobilis (Hc. mobilis) were measured at 20 K upon selective excitation at 668, 793, 810, and 815 nm with a 5 nm spectral bandwidth. When excited at 793 nm, the spectral equilibration of excitations from shorter to longer wavelength-absorbing pigments occurred within 3 ps and mostly localized at the band centered around 808 nm. When excited at 668 nm, the excitation energy transfer from the 670 nm absorbing pigment to the Qy band of BChl g took less than 0.5 ps, and the energy redistribution occurred and localized at 808 nm as in the case of the 793 nm excitation. All of the excitations were localized at the long wavelength pigment pool centered around 810 or 813 nm when excited at 810 or 815 nm. A slower energy transfer process with a time constant of 15 ps was also observed within the pool of long wavelength-absorbing pigments upon selective excitation at different wavelengths as has been observed by Lin et al. (Biophys. J. 1994, 67, 2479) when excited at 590 nm. Energy transfer from long wavelength antenna molecules to the primary electron donor P798 followed by the formation of P+ took place with a time constant of 55-70 ps for all excitations. Direct excitation of the primary electron acceptor A0, which absorbed at 670 nm, showed the same kinetic behavior as in the case when different forms of antenna pigments were excited in the Qy region. This observation generally supports the trapping-limited case of energy transfer in which the excitations have high escape probability from the reaction center (RC) until the charge separation takes place. Possible mechanisms to account for the apparent "uphill" energy transfer from the long wavelength antenna pigments to P798 are discussed.     

Probing the effect of the binding site on the electrostatic behavior of a series of carotenoids reconstituted into the light-harvesting 1 complex from purple photosynthetic bacterium Rhodospirillum rubrum detected by stark spectroscopy

Reconstitutions of the LH1 complexes from the purple photosynthetic bacterium Rhodospirillum rubrum S1 were performed with a range of carotenoid molecules having different numbers of C=C conjugated double bonds. Since, as we showed previously, some of the added carotenoids tended to aggregate and then to remain with the reconstituted LH1 complexes (Nakagawa, K.; Suzuki, S.; Fujii, R.; Gardiner, A.T.; Cogdell, R.J.; Nango, M.; Hashimoto, H. Photosynth. Res. 2008, 95, 339-344), a further purification step using a sucrose density gradient centrifugation was introduced to improve purity of the final reconstituted sample. The measured absorption, fluorescence-excitation, and Stark spectra of the LH1 complex reconstituted with spirilloxanthin were identical with those obtained with the native, spirilloxanthin-containing, LH1 complex of Rs. rubrum S1. This shows that the electrostatic environments surrounding the carotenoid and bacteriochlorophyll a (BChl a) molecules in both of these LH1 complexes were essentially the same. In the LH1 complexes reconstituted with either rhodopin or spheroidene, however, the wavelength maximum at the BChl a Qy absorption band was slightly different to that of the native LH1 complexes. These differences in the transition energy of the BChl a Qy absorption band can be explained using the values of the nonlinear optical parameters of this absorption band, i.e., the polarizability change Tr(Deltaalpha) and the static dipole-moment change |Deltamu| upon photoexcitation, as determined using Stark spectroscopy. The local electric field around the BChl a in the native LH1 complex (ES) was determined to be approximately 3.0x10(6) V/cm. Furthermore, on the basis of the values of the nonlinear optical parameters of the carotenoids in the reconstituted LH1 complexes, it is possible to suggest that the conformations of carotenoids, anhydrorhodovibrin and spheroidene, in the LH1 complex were similar to that of rhodopin glucoside in crystal structure of the LH2 complex from Rhodopseudomonas acidophila 10050.     

Mechanism of carotenoid singlet excited state energy transfer in modified bacterial reaction centers

Ultrafast transient laser spectroscopy has been used to investigate carotenoid singlet excited state energy transfer in various Rhodobacter (Rb.) sphaeroides reaction centers (RCs) modified either genetically or chemically. The pathway and efficiency of energy transfer were examined as a function of the structures and energies of the donor and acceptor molecules. On the donor side, carotenoids with various extents of pi-electron conjugation were examined. RCs studied include those from the anaerobically grown wild-type strain containing the carotenoid spheroidene, which has 10 conjugated carbon-carbon double bonds; the GA strain containing neurosporene, which has nine conjugated double bonds; and aerobically grown wild-type cells, as well as aerobically grown H(M182)L mutant, both containing the carbonyl-containing carotenoid spheroidenone, which has 11 conjugated double bonds. By varying the structure of the carotenoid, we observed the effect of altering the energies of the carotenoid excited states on the rate of energy transfer. Both S(1)- and S(2)-mediated carotenoid-to-bacteriochlorophyll energy transfer processes were observed. The highest transfer efficiency, from both the S(1) and S(2) states, was observed using the carotenoid with the shortest chain. The S(1)-mediated carotenoid-to- bacteriochlorophyll energy transfer efficiencies were determined to be 96%, 84%, and 73% for neurosporene, spheroidene, and spheroidenone, respectively. The S(2)-mediated energy transfer efficiencies follow the same trend but could not be determined quantitatively because of limitations in the time resolution of the instrumentation. The dependence of the energy transfer rate on the energetics of the energy transfer acceptor was verified by performing measurements with RCs from the H(M182)L mutant. In this mutant, the bacteriochlorophyll (denoted B(B)) located between the carotenoid and the RC special pair (P) is replaced by a bacteriopheophytin (denoted phi(B)), where the Q(X) and Q(Y) bands of phi(B) are 1830 and 1290 cm(-1), respectively, higher in energy than those of B(B). These band shifts associated with phi(B) in the H(M182)L mutant significantly alter the spectral overlap between the carotenoid and phi(B), resulting in a significant decrease of the transfer efficiency from the carotenoid S(1) state to phi(B). This leaves energy transfer from the carotenoid S(2) state to phi(B) as the dominant channel. Largely because of this change in mechanism, the overall efficiency of energy transfer from the carotenoid to P decreases to less than 50% in this mutant. Because the spectral signature of phi(B) is different from that of B(A) in this mutant, we were able to demonstrate clearly that the carotenoid-to-P energy transfer is via phi(B). This finding supports the concept that, in wild-type RCs, the carotenoid-to-P energy transfer occurs through the cofactor located at the B(B) position.     

Electron transfer in the reaction center of the Rb. sphaeroides R-26 studied by transient absorption

Electron transfer at the reaction center of the purple photosynthetic bacterium Rb. sphaeroides R-26 was measured at room temperature by the time-resolved transient absorption spectroscopy technique with 200 fs temporal resolution. The absorbance changes characteristic of the excited state of the primary donor and extending over the whole spectral range investigated from 350 nm up to 720 nm appeared after excitation with a laser pulse of about 100 fs duration at 800 nm. The time evolution of the spectra reflected the excitation of bacteriochlorophylls (BChl) M and L and the subsequent transfer of this excitation to the primary electron donor (P), with the time constant shorter than 1 ps. The decay time constant of the excited primary donor P was determined as about 3 ps, and it was faster than the rise of the reduced intermediary acceptor bacteriopheophytin (BPhe(L)). Photoreduction of BPhe(L) and its further reoxidation was clearly observed as an increase in its bleaching band intensity at around 540 nm in about 4 ps and its decrease in about 200 ps. Our findings support the theoretical model assuming the involvement of the intermediate state P(+)BChl- in the so-called "two-step" model. In this model an electron is transferred in a sequence from the excited special pair P* to bacteriochlorophyll, BChl(L), then to bacteriopheophytin, BPhe(L), and further on to quinone, Q(A). The branched charge separation, partially via P and partially via BChl(L), was also observed.     

Characterisation of uniformly 13C, 15N labelled bacteriochlorophyll a and bacteriopheophytin a in solution and in solid state: complete assignment of the 13C, 1H and 15N chemical shifts

In this investigation we report a complete assignment of (13)C, (1)H and (15)N solution and solid state chemical shifts of two bacterial photosynthetic pigments, bacteriochlorophyll (BChl) a and bacteriopheophytin (BPheo) a. Uniform stable-isotope labelling strategies were developed and applied to biosynthetic preparation of photosynthetic pigments, namely uniformly (13)C, (15)N labelled BChl a and BPheo a. Uniform stable-isotope labelling with (13)C, (15)N allowed performing the assignment of the (13)C, (15)N and (1)H resonances. The photosynthetic pigments were isolated from the biomass of photosynthetic bacteria Rhodopseudomonas palustris 17001 grown in uniformly (13)C (99%) and (15)N (98%) enriched medium. Both pigments were characterised by NMR in solution (acetone-d(6)) and by MAS NMR in solid state and their NMR resonances were recorded and assigned through standard liquid 2D (13)C-(13)C COSY, (1)H-(13)C HMQC, (1)H-(15)N HMBC and solid 2D (13)C-(13)C RFDR, (1)H-(13)C FSLG HETCOR and (1)H-(15)N HETCOR correlation techniques at 600 MHz and 750 MHz. The characterisation of pigments is of interest from biochemical to pharmaceutical industries, photosynthesis and food research.     

Intermolecular vibrational coherence in bacteriochlorophyll a with clustered polar solvent molecules

We show that resonant impulsive excitation of the Qy absorption band of bacteriochlorophyll a (BChl) launches a rapidly damped (gamma < 200 fs) ground-state coherent wave-packet motion that arises from intermolecular modes with clustered solvent molecules. Femtosecond pump-probe, dynamic-absorption signals were obtained at room temperature with BChl solutions in pyridine, acetone, and 1-propanol. The vibrational coherence observed in the 0-800-fs regime is modeled in the time domain by two (or three, in the case of 1-propanol) modulation components with asymmetric, inhomogeneously broadened line shapes and frequencies in the 100-200-cm(-1) range. The mean frequency of the vibrational coherence exhibits at least a quadratic dependence on the dipole moment of the solvent molecules and a y-intercept in the 100-cm(-1) regime. This trend is modeled by an expression for the natural frequency of a "6-12" potential composed of attractive terms from van der Waals forces and a repulsive term from the exchange (Pauli exclusion) force. The model suggests that comparable contributions to the potential are provided by the dipole-dipole and London dispersion interactions. These results support the hypothesis that the low-frequency vibrational modes in the 100-cm(-1) regime that are coupled to the light-driven charge-separation reactions in the reaction center from purple bacteria are derived from intermolecular vibrational modes between the chromophores and the surrounding protein medium.