Extension of nature's NIR-I chromophore into the NIR-II region.

The development of chromophores that absorb in the near-infrared (NIR) region beyond 1000 nm underpins numerous applications in medical and energy sciences, yet also presents substantial challenges to molecular design and chemical synthesis. Here, the core bacteriochlorin chromophore of nature's NIR absorbers, bacteriochlorophylls, has been adapted and tailored by annulation in an effort to achieve absorption in the NIR-II region. The resulting bacteriochlorin, Phen2,1-BC, contains two annulated naphthalene groups spanning meso,ß-positions of the bacteriochlorin and the 1,2-positions of the naphthalene. Phen2,1-BC was prepared via a new synthetic route. Phen2,1-BC is an isomer of previously examined Phen-BC, which differs only in attachment via the 1,8-positions of the naphthalene. Despite identical π-systems, the two bacteriochlorins have distinct spectroscopic and photophysical features. Phen-BC has long-wavelength absorption maximum (912 nm), oscillator strength (1.0), and S1 excited-state lifetime (150 ps) much different than Phen2,1-BC (1292 nm, 0.23, and 0.4 ps, respectively). These two molecules and an analogue with intermediate characteristics bearing annulated phenyl rings have unexpected properties relative to those of non-annulated counterparts. Understanding the distinctions requires extending concepts beyond the four-orbital-model description of tetrapyrrole spectroscopic features. In particular, a reduction in symmetry resulting from annulation results in electronic mixing of x- and y-polarized transitions/states, as well as vibronic coupling that together reduce oscillator strength of the long-wavelength absorption manifold and shorten the S1 excited-state lifetime. Collectively, the results suggest a heuristic for the molecular design of tetrapyrrole chromophores for deep penetration into the relatively unutilized NIR-II region.

Efficient two-step excitation energy transfer in artificial light-harvesting antenna based on bacteriochlorophyll aggregates.

Chlorosomes of green photosynthetic bacteria are large light-harvesting complexes enabling these organisms to survive at extremely low-light conditions. Bacteriochlorophylls found in chlorosomes self-organize and are ideal candidates for use in biomimetic light-harvesting in artificial photosynthesis and other applications for solar energy utilization. Here we report on the construction and characterization of an artificial antenna consisting of bacteriochlorophyll c co-aggregated with ß-carotene, which is used to extend the light-harvesting spectral range, and bacteriochlorophyll a, which acts as a final acceptor for excitation energy. Efficient energy transfer between all three components was observed by means of fluorescence spectroscopy. The efficiency varies with the ß-carotene content, which increases the average distance between the donor and acceptor in both energy transfer steps. The efficiency ranges from 89 to 37% for the transfer from ß-carotene to bacteriochlorophyll c, and from 93 to 69% for the bacteriochlorophyll c to bacteriochlorophyll a step. A significant part of this study was dedicated to a development of methods for determination of energy transfer efficiency. These methods may be applied also for study of chlorosomes and other pigment complexes.

Quenching of bacteriochlorophyll a triplet state by carotenoids in the chlorosome baseplate of green bacterium Chloroflexus aurantiacus.

To capture weak light fluxes, green photosynthetic bacteria have unique structures - chlorosomes, consisting of 104-5 molecules of bacteriochlorophyll (BChl) c, d, e. Chlorosomes are attached to the cytoplasmic membrane through the baseplate, a paracrystalline protein structure containing BChl a and carotenoids (Car). The most important function of Car is the quenching of triplet states of BChl, which prevents the formation of singlet oxygen and thereby provides photoprotection. In our work, we studied the dynamics of the triplet states of BChl a and Car in the baseplate of Chloroflexus aurantiacus chlorosomes using picosecond differential spectroscopy. BChl a of the baseplate was excited into the Qy band at 810 nm, and the corresponding absorption changes were recorded in the range of 420-880 nm. It was found that the formation of the Car triplet state occurs in ∼1.3 ns, which is ∼3 times faster than the formation of this state in the peripheral antenna of C. aurantiacus according to literature data. The Car triplet state was recorded by the characteristic absorption band T1 → Tn at ∼550 nm. Simultaneously with the appearance of absorption T1 → Tn, there was a bleaching of the singlet absorption of Car in the region of 400-500 nm. Theoretical modeling made it possible to estimate the characteristic time of formation of the triplet state of BChl a as ∼0.5 ns. It is shown that the experimental data are well described by the sequential scheme of formation and quenching of the BChl a triplet state: BChl a* → BChl aT → CarT. Thus, carotenoids from green bacteria effectively protect the baseplate from possible damage by singlet oxygen.

Experimental and Theoretical Mutation of Exciton States on the Smallest Type-I Photosynthetic Reaction Center Complex of a Green Sulfur Bacterium Chlorobaclum tepidum.

The exciton states on the smallest type-I photosynthetic reaction center complex of a green sulfur bacterium Chlorobaculum tepidum (GsbRC) consisting of 26 bacteriochlorophylls a (BChl a) and four chlorophylls a (Chl a) located on the homodimer of two PscA reaction center polypeptides were investigated. This analysis involved the study of exciton states through a combination of theoretical modeling and the genetic removal of BChl a pigments at eight sites. (1) A theoretical model of the pigment assembly exciton state on GsbRC was constructed using Poisson TrESP (P-TrESP) and charge density coupling (CDC) methods based on structural information. The model reproduced the experimentally obtained absorption spectrum, circular dichroism spectrum, and excitation transfer dynamics, as well as explained the effects of mutation. (2) Eight BChl a molecules at different locations on the GsbRC were selectively removed by genetic exchange of the His residue, which ligates the central Mg atom of BChl a, with the Leu residue on either one or two PscAs in the RC. His locations are conserved among all type-I RC plant polypeptide, cyanobacteria, and bacteria amino acid sequences. (3) Purified mutant-GsbRCs demonstrated distinct absorption and fluorescence spectra at 77 K, which were different from each other, suggesting successful pigment removal. (4) The same mutations were applied to the constructed theoretical model to analyze the outcomes of these mutations. (5) The combination of theoretical predictions and experimental mutations based on structural information is a new tool for studying the function and evolution of photosynthetic reaction centers.

Carotenoid-Mediated Long-Range Energy Transfer in the Light Harvesting-Reaction Center Complex from Photosynthetic Bacterium Roseiflexus castenholzii.

The light harvesting-reaction center complex (LH-RC) of Roseiflexus castenholzii binds bacteriochlorophylls a (BChls a), B800 and B880, absorbing around 800 and 880 nm, respectively. We comparatively investigated the interband excitation energy transfer (EET) dynamics of the wild-type LH-RC (wt-LH-RC) of Rfl. castenholzii and its carotenoid (Car)-less mutant (m-LH-RC) and found that Car can boost the B800 → B880 EET rate from (2.43 ps)-1 to (1.75 ps)-1, accounting for 38% acceleration of the EET process. Interestingly, photoexcitation of wt-LH-RC at 800 nm induced pronounced excitation dynamics of Car despite the insufficient photon energy for direct Car excitation, a phenomenon which is attributed to the BChl-Car exciplex 1[B800(↑↑)···Car(↓↓)]*. Such an exciplex is suggested to play an essential role in promoting the B800 → B880 EET process, as corroborated by the recently reported cryo-EM structures of wt-LH-RC and m-LH-RC. The mechanism of Car-mediated EET will be helpful to deepen the understanding of the role of Car in bacterial photosynthesis.

Electronic structure and energetics of a heterodimeric BChl g'/Chl a' special pair generated by exposure of Heliomicrobium modesticaldum to dioxygen.

Heliobacteria are anoxygenic phototrophs that have a Type I homodimeric reaction center containing bacteriochlorophyll g (BChl g). Previous experimental studies have shown that in the presence of light and dioxygen, BChl g is converted into 81-OH-chlorophyll aF (hereafter Chl aF), with an accompanying loss of light-driven charge separation. These studies suggest that the reaction center only loses the ability to transfer electrons once both BChl g' molecules of the P800 special pair have been converted to Chl aF'. The present work confirms that the partially converted BChl g'/Chl aF' special pair remains functional in samples exposed to dioxygen by demonstrating its presence using hyperfine couplings obtained from Q-band 1H ENDOR, 2D 14N HYSCORE and DFT methods. The DFT calculations of the BChl g'/BChl g' homodimeric primary donor, which are based on the recently published X-ray crystal structure, predict that the unpaired electron spin is equally delocalized over both BChl g' molecules and provide an excellent match to the experimental hyperfine couplings of the anaerobic samples. Exposure to dioxygen leads to substantial changes in the hyperfine interactions, indicative of greater localization of the unpaired electron spin. The measured hyperfine couplings are reproduced in the DFT calculations by replacing one of the BChl g' molecules of the primary donor with a Chl aF' molecule. The calculations reveal that the spin density becomes localized on BChl g' in the heterodimeric primary donor. Time-dependent DFT calculations demonstrate that conversion of either or both of the accessory BChl g molecules and/or one of the BChl g' molecules of P800 to Chl aF' results in minor effects on the energy of the charge-separated states. In contrast, if both of the BChl g' molecules of P800 are converted a large increase in the energy of the charge-separated state occurs. This suggests that the reaction center remains functional when only one half of the dimer is converted, however, conversion of both halves of the P800 dimer leads to loss of function.

Femtosecond Exciton Relaxation in Chlorosomes of the Photosynthetic Green Bacterium Chloroflexus aurantiacus.

Process of photosynthesis in the green bacteria Chloroflexus (Cfx.) aurantiacus starts from absorption of light by chlorosomes, peripheral antennas consisting of thousands of bacteriochlorophyll c (BChl c) molecules combined into oligomeric structures. In this case, the excited states are formed in BChl c, energy of which migrates along the chlorosome towards the baseplate and further to the reaction center, where the primary charge separation occurs. Energy migration is accompanied by non-radiative electronic transitions between the numerous exciton states, that is, exciton relaxation. In this work, we studied dynamics of the exciton relaxation in Cfx. aurantiacus chlorosomes using differential femtosecond spectroscopy at cryogenic temperature (80 K). Chlorosomes were excited by 20-fs light pulses at wavelengths in the range from 660 to 750 nm, and differential (light-dark) absorption kinetics were measured at a wavelength of 755 nm. Mathematical analysis of the obtained data revealed kinetic components with characteristic times of 140, 220, and 320 fs, which are responsible for exciton relaxation. As the excitation wavelength decreased, the number and relative contribution of these components increased. Theoretical modelling of the obtained data was carried out based of the cylindrical model of BChl c. Nonradiative transitions between the groups of exciton bands were described by a system of kinetic equations. The model that takes into account energy and structural disorder of chlorosomes turned out to be the most adequate.

Single-photon absorption and emission from a natural photosynthetic complex.

Photosynthesis is generally assumed to be initiated by a single photon1-3 from the Sun, which, as a weak light source, delivers at most a few tens of photons per nanometre squared per second within a chlorophyll absorption band1. Yet much experimental and theoretical work over the past 40 years has explored the events during photosynthesis subsequent to absorption of light from intense, ultrashort laser pulses2-15. Here, we use single photons to excite under ambient conditions the light-harvesting 2 (LH2) complex of the purple bacterium Rhodobacter sphaeroides, comprising B800 and B850 rings that contain 9 and 18 bacteriochlorophyll molecules, respectively. Excitation of the B800 ring leads to electronic energy transfer to the B850 ring in approximately 0.7 ps, followed by rapid B850-to-B850 energy transfer on an approximately 100-fs timescale and light emission at 850-875 nm (refs. 16-19). Using a heralded single-photon source20,21 along with coincidence counting, we establish time correlation functions for B800 excitation and B850 fluorescence emission and demonstrate that both events involve single photons. We also find that the probability distribution of the number of heralds per detected fluorescence photon supports the view that a single photon can upon absorption drive the subsequent energy transfer and fluorescence emission and hence, by extension, the primary charge separation of photosynthesis. An analytical stochastic model and a Monte Carlo numerical model capture the data, further confirming that absorption of single photons is correlated with emission of single photons in a natural light-harvesting complex.

Predicted Structure of the BciC Enzyme Catalyzing the Removal of the C132-Methoxycarbonyl Group for Biosynthesis of Chlorosomal Chlorophylls: A Mechanism for Dual Catalytic Functions of Hydrolysis and Decarboxylation inside Its Active Site.

Green photosynthetic bacteria, one of the phototrophs, have the largest and most efficient light-harvesting antenna systems, called chlorosomes. The core part of chlorosomes consists of unique bacteriochlorophyll c/d/e molecules. In the biosynthetic pathway of these molecules, a BciC enzyme catalyzes the removal of the C132-methoxycarbonyl group of chlorophyllide a. Two sequential reactions have been proposed for the BciC enzymatic demethoxycarbonylation: the BciC enzyme would catalyze the hydrolysis of the C132-methoxycarbonyl group, and the resulting carboxylic acid would be rapidly decarboxylated to generate pyrochlorophyllide a. In this study, we computationally predicted the three-dimensional structure of the BciC protein. Its active site was proposed based on structural analysis using docking simulation. In vitro enzymatic reaction assays of mutated BciC supported the prediction. The BciC enzymatic hydrolysis would be an aspartic/glutamic acid hydrolase, which involves the amino residues E85 and D180. Furthermore, Y58 and H126 might depend on stabilization and/or recognition with the substrate. Most importantly, H137 would protonate 13-C═O or deprotonate C132-COOH in the hydrolyzed product to promote decarboxylation. In conclusion, the BciC enzyme has the dual functions of hydrolysis and decarboxylation.

Conversion of B800 Bacteriochlorophyll a to 3-Acetyl Chlorophyll a in the Light-Harvesting Complex 3 by In Situ Oxidation.

The spectral features of energy donors and acceptors and the relationship between them in photosynthetic light-harvesting proteins are crucial for photofunctions of these proteins. Engineering energy donors and acceptors in light-harvesting proteins affords the means to increase our understanding of their photofunctional mechanisms. Herein, we demonstrate the conversion of energy-donating B800 bacteriochlorophyll (BChl) a to 3-acetyl chlorophyll (AcChl) a in light-harvesting complex 3 (LH3) from Rhodoblastus acidophilus by in situ oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. AcChl a in the B800 site exhibited a Qy band that was 111 nm blue-shifted with respect to BChl a in oxidized LH3. The structure of LH3 was barely influenced by the oxidation process, based on circular dichroism spectroscopy and size-exclusion chromatography evidence. In oxidized LH3, AcChl a transferred excitation energy to B820 BChl a, but the rate of excitation energy transfer (EET) was lower than in native LH3. The intracomplex EET in oxidized LH3 was slightly faster than in oxidized light-harvesting complex 2 (LH2). This difference is rationalized by an increase in overlap of the luminescence band of AcChl a with the long tail of the B820 absorption band in oxidized LH3 compared with that of the B850 band in oxidized LH2.

Four Routes to 3-(3-Methoxy-1,3-dioxopropyl)pyrrole, a Core Motif of Rings C and E in Photosynthetic Tetrapyrroles.

The photosynthetic tetrapyrroles share a common structural feature comprised of a ß-ketoester motif embedded in an exocyclic ring (ring E). As part of a total synthesis program aimed at preparing native structures and analogues, 3-(3-methoxy-1,3-dioxopropyl)pyrrole was sought. The pyrrole is a precursor to analogues of ring C and the external framework of ring E. Four routes were developed. Routes 1-3 entail a Pd-mediated coupling process of a 3-iodopyrrole with potassium methyl malonate, whereas route 4 relies on electrophilic substitution of TIPS-pyrrole with methyl malonyl chloride. Together, the four routes afford considerable latitude. A long-term objective is to gain the capacity to create chlorophylls and bacteriochlorophylls and analogues thereof by facile de novo means for diverse studies across the photosynthetic sciences.

Quantum chemical elucidation of a sevenfold symmetric bacterial antenna complex.

The light-harvesting complex 2 (LH2) of purple bacteria is one of the most studied photosynthetic antenna complexes. Its symmetric structure and ring-like bacteriochlorophyll arrangement make it an ideal system for theoreticians and spectroscopists. LH2 complexes from most bacterial species are thought to have eightfold or ninefold symmetry, but recently a sevenfold symmetric LH2 structure from the bacterium Mch. purpuratum was solved by Cryo-Electron microscopy. This LH2 also possesses unique near-infrared absorption and circular dichroism (CD) spectral properties. Here we use an atomistic strategy to elucidate the spectral properties of Mch. purpuratum LH2 and understand the differences with the most commonly studied LH2 from Rbl. acidophilus. Our strategy exploits a combination of molecular dynamics simulations, multiscale polarizable quantum mechanics/molecular mechanics calculations, and lineshape simulations. Our calculations reveal that the spectral properties of LH2 complexes are tuned by site energies and exciton couplings, which in turn depend on the structural fluctuations of the bacteriochlorophylls. Our strategy proves effective in reproducing the absorption and CD spectra of the two LH2 complexes, and in uncovering the origin of their differences. This work proves that it is possible to obtain insight into the spectral tuning strategies of purple bacteria by quantitatively simulating the spectral properties of their antenna complexes.

Low-Frequency Oscillations of Bacteriochlorophyll Oligomers in Chlorosomes of Photosynthetic Green Bacteria.

In green photosynthetic bacteria, light is absorbed by bacteriochlorophyll (BChl) c/d/e oligomers, which are located in chlorosomes - unique structures created by Nature to collect the energy of very weak light fluxes. Using coherent femtosecond spectroscopy at cryogenic temperature, we detected and studied low-frequency vibrational motions of BChl c oligomers in chlorosomes of the green bacteria Chloroflexus (Cfx.) aurantiacus. The objects of the study were chlorosomes isolated from the bacterial cultures grown under different light intensity. It was found that the Fourier spectrum of low-frequency coherent oscillations in the Qy band of BChl c oligomers depends on the light intensity used for the growth of bacteria. It turned out that the number of low-frequency vibrational modes of chlorosomes increases as illumination under which they were cultivated decreases. Also, the frequency range within which these modes are observed expands, and frequencies of the most modes change. Theoretical modeling of the obtained data and analysis of the literature led to conclusion that the structural basis of Cfx. aurantiacus chlorosomes are short linear chains of BChl c combined into more complex structures. Increase in the length of these chains in chlorosomes grown under weaker light leads to the observed changes in the spectrum of vibrations of BChl c oligomers. This increase is an effective mechanism for bacteria adaptation to changing external conditions.

A bacterium from a mountain lake harvests light using both proton-pumping xanthorhodopsins and bacteriochlorophyll-based photosystems.

Photoheterotrophic bacteria harvest light energy using either proton-pumping rhodopsins or bacteriochlorophyll (BChl)-based photosystems. The bacterium Sphingomonas glacialis AAP5 isolated from the alpine lake Gossenköllesee contains genes for both systems. Here, we show that BChl is expressed between 4°C and 22°C in the dark, whereas xanthorhodopsin is expressed only at temperatures below 16°C and in the presence of light. Thus, cells grown at low temperatures under a natural light-dark cycle contain both BChl-based photosystems and xanthorhodopsins with a nostoxanthin antenna. Flash photolysis measurements proved that both systems are photochemically active. The captured light energy is used for ATP synthesis and stimulates growth. Thus, S. glacialis AAP5 represents a chlorophototrophic and a retinalophototrophic organism. Our analyses suggest that simple xanthorhodopsin may be preferred by the cells under higher light and low temperatures, whereas larger BChl-based photosystems may perform better at lower light intensities. This indicates that the use of two systems for light harvesting may represent an evolutionary adaptation to the specific environmental conditions found in alpine lakes and other analogous ecosystems, allowing bacteria to alternate their light-harvesting machinery in response to large seasonal changes of irradiance and temperature.

Selective Excitation of Carotenoids of the Allochromatium vinosum Light-Harvesting LH2 Complexes Leads to Oxidation of Bacteriochlorophyll.

The mechanism of bacteriochlorophyll photooxidation in light-harvesting complexes of a number of purple photosynthetic bacteria when the complexes are excited into the carotenoid absorption bands remains unclear for many years. Here, using narrow-band laser illumination we measured action spectrum of this process for the spectral ranges of carotenoid and bacteriochlorophyll. It is shown that bacteriochlorophyll excitation results in almost no photooxidation of these molecules, while carotenoid excitation leads to oxidation with quantum yield of about 0,0003. Low value of the yield enabled an assumption that the studied process is initiated by the triplet states of the main carotenoids of the complexes with the number of conjugated double-bond chain length of N = 11. Interaction of these states with oxygen facilitates formation, though with low efficiency, of the excited singlet oxygen, which oxidizes bacteriochlorophylls. The carotenoid triplet states are formed in the process of the earlier studied singlet-triplet fission. The obtained results point at the necessity of reconsidering the functions of carotenoids in the light-harvesting complexes of purple bacteria.

Properties of Mutant Photosynthetic Reaction Centers of Purple Non-Sulfur Bacteria Cereibacter sphaeroides with M206 Ile→Gln Substitution.

In the structure of photosynthetic reaction center (RC) of the purple bacterium Cereibacter sphaeroides the highly conserved amino acid residue Ile-M206 is located near the bacteriochlorophyll dimer P, which is the primary electron donor, and the monomeric bacteriochlorophyll BA, which is the nearest electron acceptor. Since Ile-M206 is close to the C2-acetyl group of bacteriochlorophyll PB, the hydroxyl group of Tyr-M210, and to the C9-keto group of bacteriochlorophyll BA, as well as to the water molecule near the latter group, this site can be used for introducing mutations in order to study mechanisms of primary photochemical processes in the RC. Previously it was shown that the Ile→Glu substitution at the M204 position (analog of M206 in the RC of C. sphaeroides) in the RC of the closely related purple non-sulfur bacterium Rhodobacter capsulatus significantly affected kinetics of the P+HA- state formation, whereas the M204 Ile→Gln substitution led to the loss of BChl BA molecule from the complex structure. In the present work, it is shown that the single I(M206)Q or double I(M206)Q + F(M208)A amino acid substitutions in the RC of C. sphaeroides do not change the pigment composition and do not markedly influence redox potential of the primary electron donor. However, substitution of Ile M206 by Gln affected positions and amplitudes of the absorption bands of bacteriochlorophylls, increased lifetime of the primary electron donor P* excited state from 3.1 ps to 22 ps, and decreased quantum yield of the P+QA- state formation to 60%. These data suggest significant changes in the pigment-protein interactions in the vicinity of the primary electron donor P and the nearest electron acceptor BA. A considerable decrease was also noticed in the resistance of the mutant RC to thermal denaturation, which was more pronounced in the RC with the double substitution I(M206)Q + F(M208)A. This was likely associated with the disruption of the dense packing of the protein near bacteriochlorophylls PB and BA. Possible reasons for different effects of identical mutations on the properties of two highly homologous RCs from closely related purple non-sulfur bacteria are discussed.

Dynamic Stark effect in ß and γ carotenes induced by photoexcitation of bacteriochlorophyll c in chlorosomes from Chloroflexus aurantiacus.

Chlorosomes of green bacteria can be considered as a prototype of future artificial light-harvesting devices due to their unique property of self-assembly of a large number of bacteriochlorophyll (BChl) c/d/e molecules into compact aggregates. The presence of carotenoids (Cars) in chlorosomes is very important for photoprotection, light harvesting and structure stabilization. In this work, we studied for the first time the electrochromic band shift (Stark effect) in Cars of the phototrophic filamentous green bacterium Chloroflexus (Cfx.) aurantiacus induced by fs light excitation of the main pigment, BChl c. The high accuracy of the spectral measurements permitted us to extract a small wavy spectral feature, which, obviously, can be associated with the dynamic shift of the Car absorption band. A global analysis of spectroscopy data and theoretical modeling of absorption spectra showed that near 60% of Cars exhibited a red Stark shift of ~ 25 cm-1 and the remaining 40% exhibited a blue shift. We interpreted this finding as evidence of various orientations of Car in chlorosomes. We estimated the average value of the light-induced electric field strength in the place of Car molecules as ~ 106 V/cm and the average distance between Car and the neighboring BChl c as ~ 10 Å. We concluded that the dynamics of the Car electrochromic band shift mainly reflected the dynamics of exciton migration through the chlorosome toward the baseplate within ~ 1 ps. Our work has unambiguously shown that Cars are sensitive indicators of light-induced internal electric fields in chlorosomes.

Characterization of regioisomeric diterpenoid tails in bacteriochlorophylls produced by geranylgeranyl reductase from Halorhodospira halochloris and Blastochloris viridis.

Geranylgeranyl reductase (GGR) encoded by the bchP gene catalyzes the reductions of three unsaturated C = C double bonds (C6 = C7, C10 = C11, and C14 = C15) in a geranylgeranyl (GG) group of the esterifying moiety in 17-propionate residue of bacteriochlorophyll (BChl) molecules. It was recently reported that GGR in Halorhodospira halochloris potentially catalyzes two hydrogenations, yielding BChl with a tetrahydrogeranylgeranyl (THGG) tail. Furthermore, its engineered GGR, in which N-terminal insertion peptides characteristic for H. halochloris were deleted, performed single hydrogenation, producing BChl with a dihydrogeranylgeranyl (DHGG) tail. In some of these enzymatic reactions, it remained unclear in which order the C = C double bond in a GG group was first reduced. In this study, we demonstrated that the (variant) GGR from H. halochloris catalyzed an initial reduction of the C6 = C7 double bond to yield a 6,7-DHGG tail. The intact GGR of H. halochloris catalyzed the further hydrogenation of the C14 = C15 double bonds to give a 6,7,14,15-THGG group, whereas deleting the characteristic peptide region from the GGR suppressed the C14 = C15 reduction. We also verified that in a model bacterium, Blastochloris viridis producing standard BChl-b, the reduction of a GG to phytyl group occurred via 10,11-DHGG and 6,7,10,11-THGG. The high-performance liquid chromatographic elution profiles of BChls-a/b employed in this study are essential for identifying the regioisomeric diterpenoid tails in the BChls of phototrophic bacteria distributed in nature and elucidating GGR enzymatic reactions.

Isomerization kinetics of bacteriochlorophyll b and bacteriopheophytin b under acidic conditions.

Bacteriochlorophyll (BChl) b has a unique π-conjugation system, in which the bacteriochlorin macrocycle is conjugated with the C8-ethylidene group. This π-system is converted easily to the chlorin macrocycle. However, the effects of the central magnesium in BChl b on this conversion are unclear. In this study, the isomerization kinetics of BChl b and its demetalated pigment, bacteriopheophytin (BPhe) b, was analyzed under weakly acidic conditions. BChl b exhibited faster acid-induced isomerization than BPhe b. These results were attributed to the stabilization of a cationic intermediate, whose C8-ethylidene group is protonated, during the isomerization of BChl b compared to BPhe b because of a difference in the electron densities of the π-conjugation systems between BChl b and BPhe b. High-performance liquid chromatography analyses indicated that BChl b was primarily isomerized to 3-acetyl Chl a, followed by demetalation. The reaction order was due to the slower demetalation kinetics of metallobacteriochlorins than metallochlorins. These results will be helpful for handling unstable BChl b and BPhe b. The reaction properties of BChl b and BPhe b demonstrated here will be helpful for understanding the in vivo formation of BPhe b, which acts as the primary electron acceptor in photosynthetic reaction center complexes in BChl b-containing purple photosynthetic bacteria.

Energy transfer fluctuation observed by single-molecule spectroscopy of red-shifted bacteriochlorophyll in the homodimeric photosynthetic reaction center.

The photosynthetic reaction center of heliobacteria (hRC) is a homodimeric chromoprotein responsible for light harvesting and photoelectric conversion. The fluorescence of the hRC is radiated from a bacteriochlorophyll (Bchl) g having the lowest energy level, called red-Bchl g. The homodimeric architecture of the hRC indicates that it includes two red-Bchls g arranged symmetrically in pairs. Red-Bchl g is a fluorescent probe useful for monitoring the energy transfer network in the RC. Here, we show the fluorescence polarization dependences of two red-Bchls g, individually measured with selective excitation of chlorophyll a serving as the primary electron acceptor. The two red-Bchls g exhibit almost the same polarization dependences. Based on the polarization dependence and structural data of the hRC, we propose a candidate molecule for red-Bchl g. The fluorescence spectra of single hRCs represent the spectral heterogeneity reflecting the local conformational inhomogeneity. A time series of the fluorescence spectra indicates occasional peak shifts between blue- and red-shifted states without significant changes in the fluorescence intensity. The spectral fluctuation is interpreted to be due to the local conformational dynamics around a Bchl g mediating the energy transfer, switching the terminal energy acceptor between two red-Bchls g. In conclusion, while the energy transfer network in the RC can be perturbed by microscopic dynamics, the total energy transfer efficiency, i.e., the light-harvesting function, is rather robust. The functional robustness may be due to multiple energy transfer pathways composed of many antenna pigments in the RC.