Polychromatic solar energy conversion in pigment-protein chimeras that unite the two kingdoms of (bacterio)chlorophyll-based photosynthesis.

Natural photosynthesis can be divided between the chlorophyll-containing plants, algae and cyanobacteria that make up the oxygenic phototrophs and a diversity of bacteriochlorophyll-containing bacteria that make up the anoxygenic phototrophs. Photosynthetic light harvesting and reaction centre proteins from both kingdoms have been exploited for solar energy conversion, solar fuel synthesis and sensing technologies, but the energy harvesting abilities of these devices are limited by each protein's individual palette of pigments. In this work we demonstrate a range of genetically-encoded, self-assembling photosystems in which recombinant plant light harvesting complexes are covalently locked with reaction centres from a purple photosynthetic bacterium, producing macromolecular chimeras that display mechanisms of polychromatic solar energy harvesting and conversion. Our findings illustrate the power of a synthetic biology approach in which bottom-up construction of photosystems using naturally diverse but mechanistically complementary components can be achieved in a predictable fashion through the encoding of adaptable, plug-and-play covalent interfaces.

Phototriggered Dynamic and Biomimetic Growth of Chlorosomal Self-Aggregates.

Supramolecular polymerizations mimicking native systems, which are step-by-step constructions to form self-aggregates, were recently developed. However, a general system to successively and spontaneously form self-aggregates from monomeric species remains challenging. Here, we report a photoinduced supramolecular polymerization system as a biomimetic formation of chlorophyll aggregates which are the main light-harvesting antennas in photosynthetic green bacteria, called "chlorosomes". In this system, inert chlorophyll derivatives were UV-irradiated to gradually produce active species through deprotection. Such active monomers spontaneously assembled to form fiberlike chlorosomal self-aggregates in a similar manner as a dynamic growth of natural chlorosomal self-aggregates. The study would be useful for elucidation of the formation process of the chlorosomal aggregates and construction of other supramolecular structures in nature.

Ultrafast energy relaxation in single light-harvesting complexes.

Energy relaxation in light-harvesting complexes has been extensively studied by various ultrafast spectroscopic techniques, the fastest processes being in the sub-100-fs range. At the same time, much slower dynamics have been observed in individual complexes by single-molecule fluorescence spectroscopy (SMS). In this work, we use a pump-probe-type SMS technique to observe the ultrafast energy relaxation in single light-harvesting complexes LH2 of purple bacteria. After excitation at 800 nm, the measured relaxation time distribution of multiple complexes has a peak at 95 fs and is asymmetric, with a tail at slower relaxation times. When tuning the excitation wavelength, the distribution changes in both its shape and position. The observed behavior agrees with what is to be expected from the LH2 excited states structure. As we show by a Redfield theory calculation of the relaxation times, the distribution shape corresponds to the expected effect of Gaussian disorder of the pigment transition energies. By repeatedly measuring few individual complexes for minutes, we find that complexes sample the relaxation time distribution on a timescale of seconds. Furthermore, by comparing the distribution from a single long-lived complex with the whole ensemble, we demonstrate that, regarding the relaxation times, the ensemble can be considered ergodic. Our findings thus agree with the commonly used notion of an ensemble of identical LH2 complexes experiencing slow random fluctuations.

Chlorophyll f and chlorophyll d are produced in the cyanobacterium Chlorogloeopsis fritschii when cultured under natural light and near-infrared radiation.

We report production of chlorophyll f and chlorophyll d in the cyanobacterium Chlorogloeopsis fritschii cultured under near-infrared and natural light conditions. C. fritschii produced chlorophyll f and chlorophyll d when cultured under natural light to a high culture density in a 20 L bubble column photobioreactor. In the laboratory, the ratio of chlorophyll f to chlorophyll a changed from 1:15 under near-infrared, to an undetectable level of chlorophyll f under artificial white light. The results provide support that chlorophylls f and d are both red-light inducible chlorophylls in C. fritschii.

Light-induced conformational changes in photosynthetic reaction centers: dielectric relaxation in the vicinity of the dimer.

Conformational changes near the bacteriochlorophyll dimer induced by continuous illumination were identified in the wild type and 11 different mutants of reaction centers from Rhodobacter sphaeroides. The properties of the bacteriochlorophyll dimer, which has a different hydrogen bonding pattern with the surrounding protein in each mutant, were characterized by steady-state and transient optical spectroscopy. After illumination for 1 min, in the absence of the secondary quinone, the recovery of the charge-separated states was nearly 1 order of magnitude slower in one group of mutants including the wild type than in the mutants carrying the Leu to His mutation at the L131 position. The slower recovery was accompanied by a substantial decrease in the electrochromic absorption changes associated with the Q(y) bands of the nearby monomers during the illumination. The other set of mutants containing the Leu L131 to His substitution exhibited slightly altered electrochromic changes that decreased only half as much during the illumination as in the other family of mutants. The correlation between the recovery of the charge-separated states in the light-induced conformation and the electrochromic absorption changes suggests a dielectric relaxation of the protein that stabilizes the charge on the dimer.

Photochemistry of bacteriochlorophylls in human blood plasma: 2. Reaction mechanism investigated by product analysis and deuterium isotope effect.

Transmetalated (Pd) bacteriochlorophyll derivatives are currently being clinically tested as sensitizers for photodynamic therapy. Protocols using short delay times between injection and irradiation generate interest in the photochemistry of these pigments in the blood. Using near-infrared irradiation where these pigments absorb strongly, we have studied the mechanism of photo-oxidation in two lipoprotein fractions, low- and high-density lipoproteins, derived from human blood plasma that preferentially accumulate these pigments (Dandler et al. [2009] Photochem. Photobiol., 85, in press). Using quenchers of reactive oxygen species, and chemical reporters, in particular peroxides generated from cholesterol as an inherent component of the lipoproteins, a Type II mechanism generating singlet oxygen has been demonstrated for Pd- and Zn-bacteriopheophorbides. In homogeneous systems, accelerated bleaching in D(2)O, compared with H(2)O, supports this mechanism. An unusual deuterium isotope effect was observed, by contrast, in heterogeneous amphiphilic-water systems. In the early phase, and under high oxygen concentrations, again a positive D-isotope effect is observed which later, in a second phase, is reversed to a negative D-isotope effect. The latter cannot be explained by heterogeneous pigment populations in the amphiphilic system; we, therefore, conclude a mechanistic switch, and discuss a possible mechanism.

Photochemistry of bacteriochlorophylls in human blood plasma: 1. Pigment stability and light-induced modifications of lipoproteins.

Transmetalated derivatives of bacteriochlorophyll are promising sensitizers in photodynamic therapy. Protocols using short delay times between injection and irradiation cause interest in the photochemistry of these pigments in the blood. Using near-infrared irradiation where these pigments absorb strongly, we have studied the photochemistry of Zn- and Pd-bacteriopheophorbide (WST09), and of the highly polar taurinated Pd-derivative, WST11, in isolated fractions of human blood plasma. The stability of all pigments is increased in blood plasma, compared with monomeric solutions. Pd-bacteriopheophorbide is much more stable than the other two derivatives. It also has a higher capacity for inducing reactive oxygen species, yet the consumption of oxygen is comparable. There is furthermore evidence for photobleaching under anoxic conditions. The generation of hydroperoxides (ROOH) is faster with Pd- than with Zn-complexes; the formation of endoperoxides (ROOR'), measured as thiobarbituric acid reactive substances, is comparable with the two central metals. Formation of both ROOH and ROOR' is increased in low-density lipoproteins (LDL) compared with high-density lipoproteins (HDL), which is probably related to the higher concentration of target molecules in the former. In HDL, extensive cross-linking is induced among the apolipoproteins; judged from the electrophoretic mobility of LDL and HDL particles, there is also a gross structural change. Photosensitized cross-linking is much less pronounced with high-density proteins.

[Photodynamic therapy: principles and therapeutic indications]. / Principes et applications thérapeutiques de la photothérapie dynamique.

Photodynamic therapy consists in destroying a tumoral or a non tumoral tissue by the effect of both a photosensitizing molecule and a laser light. This simple concept has needed numerous years in order to be used in routine treatments with both photosensitizers and laser light delivered optimally. Researches in chemistry lead to new porphyrin and bacteriochlorophyl derivatives which alleviate the decrease of light absorption by endogenous molecules and in consequence allow a deeper light penetration. Short half-life of these compounds allows an easier treatment monitoring. In parallel, improvements in both laser technology and fibers allow new indications in various pathologies. First applications took place in treatment of respiratory, digestive and urologic cancers. The biggest success to date is recorded in ophthalmology with the treatment of age related macular degeneration. New approaches are explored and clinical studies are ongoing.

Prompt assessment of WST11-VTP outcome using luciferase transfected tumors enables second treatment and increase in overall therapeutic rate.

This study hypothesized that success rate assessment of vascular targeted photodynamic therapy (VTP) of solid tumors 24 h post-treatment may allow prompt administration of a second treatment in case of failure, increasing the overall success rate. Here, we show that treatment of luciferase transfected CT26-luc mouse colon carcinoma tumors in BALB/c mice by VTP with WST11 (a Pd-bacteriochlorophyll-based photosensitizer) allows fast assessment of treatment success 24 h post-treatment, using bioluminescence imaging (BLI). WST11-VTP was found to abolish luciferin bioluminescence in the treated tumors resulting in two types of responses. One, comprising 75% of the mice, signified successful outcome, presenting neither BLI signal nor tumor regrowth (24 h-90 days post-VTP). The second (the remaining 25% of the mice) signified treatment failure, presenting various levels of BLI signal with subsequent tumor regrowth (24 h-90 days). Consequently, the mice that failed the first treatment were treated again. We show that treatment success rate in both VTP sessions was identical and that the cumulative success rate of the treatment increased from 75% to over 90%. These results therefore, present a fast method of assessing VTP outcome and support the feasibility of successive multiple treatments with these sensitizers in the clinical arena. The presented methodology can also be helpful in future preclinical studies, and expedite the development of VTP drugs.

Ultrafast exciton-exciton coherent transfer in molecular aggregates and its application to light-harvesting systems.

Effects of the exciton-exciton coherence transfer (EECT) in strongly coupled molecular aggregates are investigated from the reduced time-evolution equation which we have developed to describe EECT. Starting with the nonlinear response function, we obtained explicit contributions from EECT to four-wave-mixing spectrum such as photon echo, taking into account double exciton states, static disorder, and heat-bath coupling represented by arbitrary spectral densities. By using the doorway-window picture and the projection operator technique, the transfer rates between two different electronic coherent states are obtained within a framework of cumulant expansion at high temperature. Applications of the present theory to strongly coupled B850 chlorophylls in the photosynthetic light harvesting system II (LH2) are discussed. It is shown that EECT is indispensable in properly describing ultrafast phenomena of strongly coupled molecular aggregates such as LH2 and that the EECT contribution to the two-dimensional optical spectroscopy is not negligible.

Theoretical prediction of spectral and optical properties of bacteriochlorophylls in thermally disordered LH2 antenna complexes.

A general approach for calculating spectral and optical properties of pigment-protein complexes of known atomic structure is presented. The method, that combines molecular dynamics simulations, quantum chemistry calculations, and statistical mechanical modeling, is demonstrated by calculating the absorption and circular dichroism spectra of the B800-B850 bacteriochlorophylls of the LH2 antenna complex from Rs. molischianum at room temperature. The calculated spectra are found to be in good agreement with the available experimental results. The calculations reveal that the broadening of the B800 band is mainly caused by the interactions with the polar protein environment, while the broadening of the B850 band is due to the excitonic interactions. Since it contains no fitting parameters, in principle, the proposed method can be used to predict optical spectra of arbitrary pigment-protein complexes of known structure.

Comparative study of the energy transfer kinetics in artificial BChl e aggregates containing a BChl a acceptor and BChl e-containing chlorosomes of Chlorobium phaeobacteroides.

Chlorosomes are the light-harvesting organelles of green bacteria, containing mainly special bacteriochlorophylls (BChls) carrying a 3(1)-hydroxy side chain. Artificial aggregates of BChl c, d, and e have been shown to resemble the native chlorosomes in many respects. They are therefore seen as good model systems for understanding the spectroscopic properties of these antenna systems. We have investigated the excitation energy transfer in artificial aggregates of BChl e, containing small amounts of BChl a as an energy acceptor, using steady-state and time-resolved fluorescence. Global analysis of the kinetic data yields two lifetimes attributable to energy transfer: a fast one of 12-20 ps and a slower one of approximately 50 ps. For comparison, BChl e-containing native chlorosomes of Chlorobium phaeobacteroides and chlorosomes in which the energy acceptor had been degraded by alkaline treatment were also studied. A similar behavior is seen in both the artificial and the natural systems. The results suggest that the artificial aggregates of BChls have a potential as antenna systems in future artificial photonic devices.

Photostability of bacteriochlorophyll a and derivatives: potential sensitizers for photodynamic tumor therapy.

The photostabilities of bacteriochlorophyll a and several of its derivatives, which are of interest as potential sensitizers in photodynamic tumor therapy, were investigated. The pigments were irradiated with light >630 nm in organic solvents (acetone, tetrahydrofuran, pyridine, methanol, ethanol, n-propanol, 2-propanol and toluene) and in aqueous detergent solutions (cetyl-trimethyl-ammonium bromide [CTAB], lauryldimethyl-aminoxide [LDAO] or sodium dodecyl-sulfate [SDS] and Triton X-100 [TX100]). Their stabilities in these different solvents were determined in the presence and absence of an external sensitizer (pyromethyl-pheophorbide a), oxygen, sodium ascorbate and inert gas (Ar) or vacuum. The photodegradation products of bacteriochlorophyll a in acetone solution were isolated, purified by HPLC and analyzed by their absorption spectra and mass spectroscopy. Besides the well-known dehydrogenation products, such as [3-acetyl]-chlorophyll a, which were obtained as by-products, the major products had low absorption in the visible-near infrared spectral range. The spectral signature of the major component of these products was characteristic of linear open-chain tetrapyrroles, but they lacked the characteristic protonation-deprotonation behavior and reactivity of bilins with Zn(++).

Novel water-soluble bacteriochlorophyll derivatives for vascular-targeted photodynamic therapy: synthesis, solubility, phototoxicity and the effect of serum proteins.

New negatively charged water-soluble bacteriochlorophyll (Bchl) derivatives were developed in our laboratory for vascular-targeted photodynamic therapy (VTP). Here we focused on the synthesis, characterization and interaction of the new candidates with serum proteins and particularly on the effect of serum albumin on the photocytotoxicity of WST11, a representative compound of the new derivatives. Using several approaches, we found that aminolysis of the isocyclic ring with negatively charged residues markedly increases the hydrophilicity of the Bchl sensitizers, decreases their self-association constant and selectively increases their affinity to serum albumin, compared with other serum proteins. The photocytotoxicity of the new candidates in endothelial cell culture largely depends on the concentration of the serum albumin. Importantly, after incubation with physiological concentrations of serum albumin (500-600 microM), WST11 was found to be poorly photocytotoxic (>80% endothelial cell survival in cell cultures). However, in a recent publication (Mazor, O. et al. [2005] Photochem. Photobiol. 81, 342-351) we showed that VTP of M2R melanoma xenografts with a similar WST11 concentration resulted in approximately 100% tumor flattening and >70% cure rate. We therefore propose that the two studies collectively suggest that the antitumor activity of WST11 and probably of other similar candidates does not depend on direct photointoxication of individual endothelial cells but on the vascular tissue response to the VTP insult.

The impact of different intensities of green light on the bacteriochlorophyll homologue composition of the Chlorobiaceae Prosthecochloris aestuarii and Chlorobium phaeobacteroides.

Members of the Chlorobiaceae and Chloroflexaceae are unique among the phototrophic micro-organisms in having a remarkably rich chlorophyll pigment diversity. The physiological regulation of this diversity and its ecological implications are still enigmatic. The bacteriochlorophyll composition of the chlorobiaceae Prosthecochloris aestuarii strain CE 2404 and Chlorobium phaeobacteroides strain UdG 6030 was therefore studied by both HPLC with photodiode array (PDA) detection and liquid chromatography-mass spectrometry (LC-MS). These strains were grown in liquid cultures under green light (480-615 nm) at different light intensities (0.2-55.7 micromol photons m(-2) s(-1)), simulating the irradiance regime at different depths of the water column of deep lakes. The specific growth rates of Ptc. aestuarii under green light achieved a maximum of 0.06 h(-1) at light intensities exceeding 6 micromol photons m(-2) s(-1), lower than the maximum observed under white light (approx. 0.1 h(-1)). The maximal growth rates of Chl. phaeobacteroides under green light were slightly higher (0.07 h(-1)) than observed for Ptc. aestuarii and were achieved at 3.5 and 4.3 micromol photons m(-2) s(-1). LC-MS/MS analysis of pigment extracts revealed most (>90 %) BChl c homologues of Ptc. aestuarii to be esterified with farnesol. The homologues differed in mass by multiples of 14 Da, reflecting different alkyl subsituents at positions C-8 and C-12 on the tetrapyrrole macrocycle. The relative proportions of the individual homologues varied only slightly among different light intensities. The specific content of BChl c was maximal at 3-5 micromol photons m(-2) s(-1) [400+/-150 nmol BChl c (mg protein)(-1)]. In the case of Chl. phaeobacteroides, the specific content of BChl e was maximal at 4.3 micromol photons m(-2) s(-1) [115 nmol BChl e (mg protein)(-1)], and this species was characterized by high carotenoid (isorenieratene) contents. The major BChl e forms were esterified with a range of isoprenoid and straight-chain alcohols. The major isoprenoid alcohols comprised mainly farnesol and to a lesser extent geranylgeraniol. The straight-chain alcohols included C(15), C(15 : 1), C(16), C(16 : 1) and C(17). Interestingly, the proportion of straight alkyl chains over isoprenoid esterified side chains shifted markedly with increasing light intensity: the isoprenoid side chains dominated at low light intensities, while the straight-chain alkyl substituents dominated at higher light intensities. The authors propose that this phenomenon may be explained as a result of changing availability of reducing power, i.e. the highly reduced straight-chain alcohols have a higher biosynthetic demand for NADPH(2) than the polyunsaturated isoprenoid with the same number of carbon atoms.

Diastereoselective self-aggregation of synthetic 3-(1-hydroxyethyl)-bacteriopyrochlophyll-a as a novel photosynthetic antenna model absorbing near the infrared regions.

3-Deacetyl-3-(1-hydroxyethyl)bacteriopyrochlorophyll-a (1), 7,8-dihydrobacteriochlorophyll-d possessing 8-ethyl, 12-methyl and 17(4)-phytyl groups, was prepared by modifying naturally occurring bacteriochlorophyll-a. The synthetic 3(1)-epimers were separated by high-performance liquid chromotagraphy, and the absolute configuration at the 3(1)-position was determined by derivatization of 1 to a structurally determined chlorin. A dichloromethane solution of 3(1)R-1 or 3(1)S-1 was diluted with a 1000-fold volume of cyclohexane to give self-aggregation species absorbing light at a near-infrared (NIR) region (<910 nm). The resulting Qy maximum in 3(1)R-1 was 860 nm and redshifted by 2170 cm(-1) from the monomeric one, whereas epimeric 3(1)S-1 showed a less redshifted peak at ca 800 nm, with a small dimeric band around 740 nm. Such visible spectra indicated that 3(1)R/S-1 formed different supramolecular structures in the self-aggregates. In contrast, self-aggregation of the 7,8-dehydro-compound 2, bacteriochlorophyll-dP, found in natural antennas of photosynthetic green bacteria showed much smaller diastereomeric control. The self-aggregates of 3(1)R-1 absorbing light in the NIR region would be models of intrinsic membraneous light-harvesting complexes 1 in photosynthetic purple bacteria as well as extramembranous antennas in green bacteria.

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 10(11) photons x pulse(-1) x 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 (approximately 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 ( approximately 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.