State transition is quiet around pyrenoid and LHCII phosphorylation is not essential for thylakoid deformation in Chlamydomonas 137c.

Photosynthetic organisms have developed a regulation mechanism called state transition (ST) to rapidly adjust the excitation balance between the two photosystems by light-harvesting complex II (LHCII) movement. Though many researchers have assumed coupling of the dynamic transformations of the thylakoid membrane with ST, evidence of that remains elusive. To clarify the above-mentioned coupling in a model organism Chlamydomonas, here we used two advanced microscope techniques, the excitation-spectral microscope (ESM) developed recently by us and the superresolution imaging based on structured-illumination microscopy (SIM). The ESM observation revealed ST-dependent spectral changes upon repeated ST inductions. Surprisingly, it clarified a less significant ST occurrence in the region surrounding the pyrenoid, which is a subcellular compartment specialized for the carbon-fixation reaction, than that in the other domains. Further, we found a species dependence of this phenomenon: 137c strain showed the significant intracellular inhomogeneity of ST occurrence, whereas 4A+ strain hardly did. On the other hand, the SIM observation resolved partially irreversible fine thylakoid transformations caused by the ST-inducing illumination. This fine, irreversible thylakoid transformation was also observed in the STT7 kinase-lacking mutant. This result revealed that the fine thylakoid transformation is not induced solely by the LHCII phosphorylation, suggesting the highly susceptible nature of the thylakoid ultrastructure to the photosynthetic light reactions.

Development of Line-Detected UV-Vis Absorption Microscope and Its Application to Quantitative Evaluation of Lithium Surface Reactivity.

Electrochemical reactions in practical batteries occur in confined environments where anode and cathode electrodes are separated only by a thin separator. Therefore, their electrochemical behaviors may differ from those obtained in the conventional experimental cells, where the two electrodes (working and counter electrodes) are largely separated compared to the batteries. The spatial and temporal distributions of the chemical species in the vicinity of each electrode are highly expected to be determined for quantitatively understanding the phenomena in confined environments. In the present study, we developed a line-detected UV-vis absorption microscope that simultaneously measures space-resolved UV-vis absorption spectra. This novel technique has been successfully applied to evaluate the reactivities of the highly reactive lithium (Li) surfaces in organic electrolyte solutions under in situ conditions. The quantitative evaluations of the dissolution rate of Li and the diffusion constant of the product were successfully realized by analyzing the space- and time-resolved absorption spectra based on Fick's law of diffusion. The microscopic technique is expected to open the door to understanding the fundamental electrochemistry in batteries.

Exciton quenching by oxidized chlorophyll Z across the two adjacent monomers in a photosystem II core dimer.

This study aimed to clarify (1) which pigment in a photosystem II (PSII) core complex is responsible for the 695-nm emission at 77 K and (2) the molecular basis for the oxidation-induced fluorescence quenching in PSII. Picosecond time-resolved fluorescence dynamics was compared between the dimeric and monomeric PSII with and without addition of an oxidant. The results indicated that the excitation-energy flow to the 695-nm-emitting chlorophyll (Chl) at 36 K and 77 K was hindered upon monomerization, clearly demonstrating significant exciton migration from the Chls on one monomer to the 695-nm-emitting pigment on the adjacent monomer. Oxidation of the redox-active Chl, which is named ChlZ caused almost equal quenching of the 684-nm and 695-nm emission bands in the dimer, and lower quenching of the 695-nm band in the monomer. These results suggested two possible scenarios responsible for the 695-nm emission band: (A) Chl11-13 pair and the oxidized ChlZD1 work as the 695-nm emitting Chl and the quenching site, respectively, and (B) Chl29 and the oxidized ChlZD2 work as the 695-nm emitting Chl and the quenching site, respectively.

High-Speed Excitation-Spectral Microscopy Uncovers In Situ Rearrangement of Light-Harvesting Apparatus in Chlamydomonas during State Transitions at Submicron Precision.

Photosynthetic organisms adjust to fluctuating natural light under physiological ambient conditions through flexible light-harvesting ability of light-harvesting complex II (LHCII). A process called state transition is an efficient regulation mechanism to balance the excitations between photosystem II (PSII) and photosystem I (PSI) by shuttling mobile LHCII between them. However, in situ observation of the migration of LHCII in vivo remains limited. In this study, we investigated the in vivo reversible changes in the intracellular distribution of the chlorophyll (Chl) fluorescence during the light-induced state transitions in Chlamydomonas reinhardtii. The newly developed noninvasive excitation-spectral microscope provided powerful spectral information about excitation-energy transfer between Chl-a and Chl-b. The excitation spectra were detected through the fluorescence emission in the 700-750-nm spectral range, where PSII makes the main contribution, though PSI still makes a non-negligible contribution at room temperature. The technique is sensitive to the Chl-b spectral component specifically bound to LHCII. Using a PSI-specific 685-nm component also provided visualization of the local relative concentration of PSI within a chloroplast at room temperature. The decrease in the relative intensity of the Chl-b band in state 2 was more conspicuous in the PSII-rich region than in the PSI-rich region, reflecting the dissociation of LHCII from PSII. We observed intracellular redistributions of the Chl-b-related light-harvesting abilities within a chloroplast during the state transitions. This observation implies the association of the state transitions with the morphological changes in the thylakoid membrane.

Development of a Multicolor Line-Focus Microscope for Rapid Acquisitions of Excitation Spectra.

To conduct rapid microscope observations with the excitation spectral measurement for photosynthetic organisms, a wavelength-dispersive line-focus microscope was developed. In the developed system, fluorescence signals at multiple positions on a sample excited with different wavelengths can be detected as a two-dimensional image on the EMCCD camera at the same time. Using the developed system, one can obtain excitation spectra at every pixel over the excitation wavelength range from 635 to 695 nm, which covers the full range of the Qy bands of both chlorophyll-a and chlorophyll-b. Recording the reference laser spectra at the same time ensures robust measurement against the moderate spectral fluctuation in the excitation laser. Using an objective lens with a numerical aperture of 0.9, the lateral and axial resolutions of 0.56 and 1.08 µm, respectively, were achieved. The theoretically limited and experimentally estimated spectral resolutions of the excitation spectral measurement were 0.86 and 1.3 nm, respectively. The validity of the system was demonstrated by measuring fluorescent beads and single cells of a model alga, Chlamydomonas reinhardtii. Intrachloroplast inhomogeneity in the relative intensity of the chlorophyll-b band could be visualized in Chlamydomonas cells. The inhomogeneity reflects the intrachloroplast variation in the local peripheral antenna size.

Red shift in the spectrum of a chlorophyll species is essential for the drought-induced dissipation of excess light energy in a poikilohydric moss, Bryum argenteum.

Some mosses are extremely tolerant of drought stress. Their high drought tolerance relies on their ability to effectively dissipate absorbed light energy to heat under dry conditions. The energy dissipation mechanism in a drought-tolerant moss, Bryum argenteum, has been investigated using low-temperature picosecond time-resolved fluorescence spectroscopy. The results are compared between moss thalli samples harvested in Antarctica and in Japan. Both samples show almost the same quenching properties, suggesting an identical drought tolerance mechanism for the same species with two completely different habitats. A global target analysis was applied to a large set of data on the fluorescence-quenching dynamics for the 430-nm (chlorophyll-a selective) and 460-nm (chlorophyll-b and carotenoid selective) excitations in the temperature region from 5 to 77 K. This analysis strongly suggested that the quencher is formed in the major peripheral antenna of photosystem II, whose emission spectrum is significantly broadened and red-shifted in its quenched form. Two emission components at around 717 and 725 nm were assigned to photosystem I (PS I). The former component at around 717 nm is mildly quenched and probably bound to the PS I core complex, while the latter at around 725 nm is probably bound to the light-harvesting complex. The dehydration treatment caused a blue shift of the PS I emission peak via reduction of the exciton energy flow to the pigment responsible for the 725 nm band.

Fluorescence property of photosystem II protein complexes bound to a gold nanoparticle.

Development of an efficient photo-anode system for water oxidation is key to the success of artificial photosynthesis. We previously assembled photosystem II (PSII) proteins, which are an efficient natural photocatalyst for water oxidation, on a gold nanoparticle (GNP) to prepare a PSII-GNP conjugate as an anode system in a light-driven water-splitting nano-device (Noji et al., J. Phys. Chem. Lett., 2011, 2, 2448-2452). In the current study, we characterized the fluorescence property of the PSII-GNP conjugate by static and time-resolved fluorescence measurements, and compared with that of free PSII proteins. It was shown that in a static fluorescence spectrum measured at 77 K, the amplitude of a major peak at 683 nm was significantly reduced and a red shoulder at 693 nm disappeared in PSII-GNP. Time-resolved fluorescence measurements showed that picosecond components at 683 nm decayed faster by factors of 1.4-2.1 in PSII-GNP than in free PSII, explaining the observed quenching of the major fluorescence peak. In addition, a nanosecond-decay component arising from a 'red chlorophyll' at 693 nm was lost in time-resolved fluorescence of PSII-GNP, probably due to a structural perturbation of this chlorophyll by interaction with GNP. Consistently with these fluorescence properties, degradation of PSII during strong-light illumination was two times slower in PSII-GNP than in free PSII. The enhanced durability of PSII is an advantageous property of the PSII-GNP conjugate in the development of an artificial photosynthesis device.

Development of a novel cryogenic microscope with numerical aperture of 0.9 and its application to photosynthesis research.

A novel cryogenic optical-microscope system was developed in which the objective lens is set inside of the cryostat adiabatic vacuum space. Being isolated from the sample when it was cooled, the objective lens was maintained at room temperature during the cryogenic measurement. Therefore, the authors were able to use a color-aberration corrected objective lens with a numerical aperture of 0.9. The lens is equipped with an air vent for compatibility to the vacuum. The theoretically expected spatial resolutions of 0.39µm along the lateral direction and 1.3µm along the axial direction were achieved by the developed system. The system was applied to the observations of non-uniform distributions of the photosystems in the cells of a green alga, Chlamydomonas reinhardtii, at 94K. Gaussian decomposition analysis of the fluorescence spectra at all the pixels clearly demonstrated a non-uniform distribution of the two photosystems, as reflected in the variable ratios of the fluorescence intensities assigned to photosystem II and to those assigned to photosystem I. The system was also applied to the fluorescence spectroscopy of single isolated photosystem I complexes at 90K. The fluorescence, assigned to be emitted from a single photosystem I trimer, showed an intermittent fluctuation called blinking, which is typical for a fluorescence signal from a single molecule. The vibronic fluorescence bands at around 790nm were observed for single photosystem I trimers, suggesting that the color aberration is not serious up to the 800nm spectral region.

Study of cell-differentiation and assembly of photosynthetic proteins during greening of etiolated Zea mays leaves using confocal fluorescence microspectroscopy at liquid-nitrogen temperature.

Fluorescence microspectroscopy observations were used to study the processes of cell differentiation and assemblies of photosynthesis proteins in Zea mays leaves under the greening process. The observations were done at 78K by setting the sample in a cryostat to avoid any undesired progress of the greening process during the measurements. The lateral and axial spatial resolutions of the system were 0.64µm and 4.4µm, respectively. The study revealed the spatial distributions of protochlorophyllide (PChld) in both the 632-nm-emitting and 655-nm-emitting forms within etiolated Zea mays leaves. The sizes of the fluorescence spots attributed to the former were larger than those of the latter, validating the assignment of the former and latter to the prothylakoid and prolamellar bodies, respectively. In vivo microspectroscopy observations of mature Zea mays leaves confirmed the different photosystem II (PS I)/photosystem I (PS II) ratio between the bundle sheath (BS) and mesophyll (MS) cells, which is specific for C4-plants. The BS cells in Zea mays leaves 1h after the initiation of the greening process tended to show fluorescence spectra at shorter wavelength side (at around 679nm) than the MS cells (at around 682nm). The 679-nm-emitting chlorophyll-a form observed mainly in the BS cells was attributed to putative precursor complexes to PS I. The BS cells under 3-h greening showed higher relative intensities of the PS I fluorescence band at around 735nm, suggesting the reduced PS II amount in the BS cells in this greening stage.

Two types of fucoxanthin-chlorophyll-binding proteins I tightly bound to the photosystem I core complex in marine centric diatoms.

Intact fucoxanthin (Fucox)-chlorophyll (Chl)-binding protein I-photosystem I supercomplexes (FCPI-PSIs) were prepared by a newly developed simple fast procedure from centric diatoms Chaetoceros gracilis and Thalassiosira pseudonana to study the mechanism of their efficient solar energy accumulation. FCPI-PSI purified from C. gracilis contained 252 Chl a, 23 Chl c, 56 Fucox, 34 diadinoxanthin+diatoxanthin, 1 violaxanthin, 21 ß-carotene, and 2 menaquinone-4 per P700. The complex showed a high electron transfer activity at 185,000µmolmg Chl a(-1)·h(-1) to reduce methyl viologen from added cytochrome c6. We identified 14 and 21 FCP proteins in FCPI-PSI of C. gracilis and T. pseudonana, respectively, determined by N-terminal and internal amino acid sequences and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. PsaO and a red lineage Chla/b-binding-like protein (RedCAP), Thaps3:270215, were also identified. Severe detergent treatment of FCPI-PSI released FCPI-1 first, leaving the FCPI-2-PSI-core complex. FCPI-1 contained more Chl c and showed Chl a fluorescence at a shorter wavelength than FCPI-2, suggesting an excitation-energy transfer from FCPI-1 to FCPI-2 and then to the PSI core. Fluorescence emission spectra at 17K in FCPI-2 varied depending on the excitation wavelength, suggesting two independent energy transfer routes. We formulated a model of FCPI-PSI based on the biochemical assay results.

Access to the Antenna System of Photosystem I via Single-Molecule Excitation-Emission Spectroscopy.

In the development of single-molecule spectroscopy, the simultaneous detection of the excitation and emission spectra has been limited. The fluorescence excitation spectrum based on background-free signals is compatible with the fluorescence-emission-based detection of single molecules and can provide insight into the variations in the input energy of the different terminal emitters. Here, we implement single-molecule excitation-emission spectroscopy (SMEES) for photosystem I (PSI) via a cryogenic optical microscope. To this end, we extended our line-focus-based excitation-spectral microscope system to the cryogenic temperature-compatible version. PSI is one of the two photosystems embedded in the thylakoid membrane in oxygen-free photosynthetic organisms. PSI plays an essential role in electron transfer in the photosynthesis reaction. PSIs of many organisms contain a few red-shifted chlorophylls (Chls) with much lower excitation energies than ordinary antenna Chls. The fluorescence emission spectrum originates primarily from the red-shifted Chls, whereas the excitation spectrum is sensitive to the antenna Chls that are upstream of red-shifted Chls. Using SMEES, we obtained the inclining two-dimensional excitation-emission matrix (2D-EEM) of PSI particles isolated from a cyanobacterium, Thermosynechococcus vestitus (equivalent to elongatus), at about 80 K. Interestingly, by decomposing the inclining 2D-EEMs within time course observation, we found prominent variations in the excitation spectra of the red-shifted Chl pools with different emission wavelengths, strongly indicating the variable excitation energy transfer (EET) pathway from the antenna to the terminal emitting pools. SMEES helps us to directly gain information about the antenna system, which is fundamental to depicting the EET within pigment-protein complexes.

Photosystem II does not possess a simple excitation energy funnel: time-resolved fluorescence spectroscopy meets theory.

The experimentally obtained time-resolved fluorescence spectra of photosystem II (PS II) core complexes, purified from a thermophilic cyanobacterium Thermosynechococcus vulcanus, at 5-180 K are compared with simulations. Dynamic localization effects of excitons are treated implicitly by introducing exciton domains of strongly coupled pigments. Exciton relaxations within a domain and exciton transfers between domains are treated on the basis of Redfield theory and generalized Förster theory, respectively. The excitonic couplings between the pigments are calculated by a quantum chemical/electrostatic method (Poisson-TrEsp). Starting with previously published values, a refined set of site energies of the pigments is obtained through optimization cycles of the fits of stationary optical spectra of PS II. Satisfactorily agreement between the experimental and simulated spectra is obtained for the absorption spectrum including its temperature dependence and the linear dichroism spectrum of PS II core complexes (PS II-CC). Furthermore, the refined site energies well reproduce the temperature dependence of the time-resolved fluorescence spectrum of PS II-CC, which is characterized by the emergence of a 695 nm fluorescence peak upon cooling down to 77 K and the decrease of its relative intensity upon further cooling below 77 K. The blue shift of the fluorescence band upon cooling below 77 K is explained by the existence of two red-shifted chlorophyll pools emitting at around 685 and 695 nm. The former pool is assigned to Chl45 or Chl43 in CP43 (Chl numbering according to the nomenclature of Loll et al. Nature2005, 438, 1040) while the latter is assigned to Chl29 in CP47. The 695 nm emitting chlorophyll is suggested to attract excitations from the peripheral light-harvesting complexes and might also be involved in photoprotection.

Arabitol provided by lichenous fungi enhances ability to dissipate excess light energy in a symbiotic green alga under desiccation.

Lichens are drought-resistant symbiotic organisms of mycobiont fungi and photobiont green algae or cyanobacteria, and have an efficient mechanism to dissipate excess captured light energy into heat in a picosecond time range to avoid photoinhibition. This mechanism can be assessed as drought-induced non-photochemical quenching (d-NPQ) using time-resolved fluorescence spectroscopy. A green alga Trebouxia sp., which lives within a lichen Ramalina yasudae, is one of the most common green algal photobionts. This alga showed very efficient d-NPQ under desiccation within the lichen thallus, whereas it lost d-NPQ ability when isolated from R. yasudae, indicating the importance of the interaction with the mycobiont for d-NPQ ability. We analyzed the water extracts from lichen thalli that enhanced d-NPQ in Trebouxia. Of several sugar compounds identified in the water extracts by nuclear magnetic resonance (NMR), mass spectrometry (MS) and gas chromatography (GC) analyses, only d-arabitol recovered d-NPQ in isolated Trebouxia to a level similar to that detected for R. yasudae thallus. Other sugar compounds did not help the expression of d-NPQ at the same concentrations. Thus, arabitol is essential for the expression of d-NPQ to dissipate excess captured light energy into heat, protecting the photobiont from photoinhibition. The relationship between mycobionts and photobionts is, therefore, not commensalism, but mutualism with each other, as shown by d-NPQ expression.

Uphill energy transfer mechanism for photosynthesis in an Antarctic alga.

Prasiola crispa, an aerial green alga, forms layered colonies under the severe terrestrial conditions of Antarctica. Since only far-red light is available at a deep layer of the colony, P. crispa has evolved a molecular system for photosystem II (PSII) excitation using far-red light with uphill energy transfer. However, the molecular basis underlying this system remains elusive. Here, we purified a light-harvesting chlorophyll (Chl)-binding protein complex from P. crispa (Pc-frLHC) that excites PSII with far-red light and revealed its ring-shaped structure with undecameric 11-fold symmetry at 3.13 Šresolution. The primary structure suggests that Pc-frLHC evolved from LHCI rather than LHCII. The circular arrangement of the Pc-frLHC subunits is unique among eukaryote LHCs and forms unprecedented Chl pentamers at every subunit‒subunit interface near the excitation energy exit sites. The Chl pentamers probably contribute to far-red light absorption. Pc-frLHC's unique Chl arrangement likely promotes PSII excitation with entropy-driven uphill excitation energy transfer.

Supramolecular energy transfer from photoexcited chlorosomal zinc porphyrin self-aggregates to a chlorin or bacteriochlorin monomer as models of main light-harvesting antenna systems in green photosynthetic bacteria.

Self-aggregates of a synthetic zinc porphyrin worked as a light absorber and photoexcited energy donor, transferred the collected energy to a small amount of 3-acetyl-(bacterio)chlorin monomer, and induced near-infrared fluorescence from the acceptors in aqueous micellar solution. These artificial supramolecular systems are novel models of the main light-harvesting antennas of green photosynthetic bacteria, chlorosomes.

Accumulation of quenched LHCII around PSI in Chlamydomonas cells in state2 revealed by cryo-fluorescence lifetime imaging.

Fluorescence-spectral microscope observations of photosynthetic organisms at cryogenic temperatures have the ability to spectrally resolve the two photosystems (PSs) and thus provide a powerful tool to elucidate the functional analysis of photosynthesis in vivo. In the present study, a measurement channel of the fluorescence lifetime at 680 nm was added to the cryo-microscope system previously developed by the authors. This provides access to information on the functional state of the light-harvesting system in living cells during regulation by a mechanism called state transitions. The observations of state1-locked and state2-locked Chlamydomonas cells at 80 K enabled us to identify a component showing rapidly decaying fluorescence with a lifetime of ca. 3 ps and emitting at around 676 nm. The component was assigned to the light-harvesting complex II (LHCII) that is isolated from both PSs and in a quenched state, probably due to the formation of aggregates. Simultaneous spectral observations revealed the accumulation of this free LHCII in the photosystem I (PSI)-enriched region within each state2-locked cell. To the best of our knowledge, this is the first in-vivo observation which suggests the localization of the quenched LHCII aggregates.

Mechanism of strong quenching of photosystem II chlorophyll fluorescence under drought stress in a lichen, Physciella melanchla, studied by subpicosecond fluorescence spectroscopy.

The mechanism of the severe quenching of chlorophyll (Chl) fluorescence under drought stress was studied in a lichen Physciella melanchla, which contains a photobiont green alga, Trebouxia sp., using a streak camera and a reflection-mode fluorescence up-conversion system. We detected a large 0.31 ps rise of fluorescence at 715 and 740 nm in the dry lichen suggesting the rapid energy influx to the 715-740 nm bands from the shorter-wavelength Chls with a small contribution from the internal conversion from Soret bands. The fluorescence, then, decayed with time constants of 23 and 112 ps, suggesting the rapid dissipation into heat through the quencher. The result confirms the accelerated 40 ps decay of fluorescence reported in another lichen (Veerman et al., 2007 [36]) and gives a direct evidence for the rapid energy transfer from bulk Chls to the longer-wavelength quencher. We simulated the entire PS II fluorescence kinetics by a global analysis and estimated the 20.2 ns(-1) or 55.0 ns(-1) energy transfer rate to the quencher that is connected either to the LHC II or to the PS II core antenna. The strong quenching with the 3-12 times higher rate compared to the reported NPQ rate, suggests the operation of a new type of quenching, such as the extreme case of Chl-aggregation in LHCII or a new type of quenching in PS II core antenna in dry lichens.

Multiple dissipation components of excess light energy in dry lichen revealed by ultrafast fluorescence study at 5 K.

A time-resolved fluorescence study of living lichen thalli at 5 K was conducted to clarify the dynamics and mechanism of the effective dissipation of excess light energy taking place in lichen under extreme drought conditions. The decay-associated spectra obtained from the experiment at 5 K were characterized by a drastically sharpened spectral band which could not be resolved by experiments at higher temperatures. The present results indicated the existence of two distinct dissipation components of excess light energy in desiccated lichen; one is characterized as rapid fluorescence decay with a time constant of 27 ps in the far-red region that was absent in wet lichen thalli, and the other is recognized as accelerated fluorescence decay in the 685-700 nm spectral region. The former energy-dissipation component with extremely high quenching efficiency is most probably ascribed to the emergence of a rapid quenching state in the peripheral-antenna system of photosystem II (PS II) on desiccation. This is an extremely effective protection mechanism of PS II under desiccation, which lichens have developed to survive in the severely desiccated environments. The latter, which is less efficient at 5 K, might have a supplementary role and take place either in the core antenna of PS II or aggregated peripheral antenna of PS II.

Intensity borrowing via excitonic couplings among soret and Q(y) transitions of bacteriochlorophylls in the pigment aggregates of chlorosomes, the light-harvesting antennae of green sulfur bacteria.

Model calculations of linear dichroism (LD) and circular dichroism (CD) spectra were conducted for the chlorosomes of green sulfur bacteria, Chlorobium phaeobacteroides and Chlorobium tepidum, on the basis of the theory of delocalized exciton. The chlorosomes of Chl. phaeobacteroides and Chl. tepidum contain bacteriochlorophylls (BChls) e and c as the major light-harvesting pigments, respectively. The excitonic couplings among the Soret and Q(y) transitions were considered on the basis of the "rodlike" structural model for the pigment self-aggregates in a chlorosome. Trial simulations were conducted by assuming the B(x) and B(y) transition-dipole vectors to be parallel to the molecular x- and y-axes, respectively. The simulation at this stage could nicely reproduce the larger splitting of the Soret band and more significant enhancement of the Q(y) band upon formation of chlorosome in the BChl e-containing chlorosome than in the BChl c-containing one. Intensity borrowing was indicated to be the key mechanism inducing the enhancement of the Q(y) band in the BChl e-containing chlorosomes. However, the simulated LD and CD spectra in the Soret region showed qualitative disagreement from the observed ones. To resolve the deviations, the directions of the B(x) and B(y) transition-dipole vectors and the orientations of the molecular planes of BChls were adjusted in the next stage. The fine-tuning of these parameters resulted in a striking agreement between the observed and simulated CD and LD spectra over the whole spectral range studied. The best fit was obtained when the B(x) and B(y) transition-dipole vectors were assumed to be rotated 25 degrees clockwise from the molecular x- and y-axes and the molecular planes in the pigment aggregate were tilted 5 degrees from that assumed in the original model without alteration in the direction of the molecular y-axis. The calculated spectral profiles were affected little by the change in the curvatures of the rod surface, showing that the optical spectra of chlorosomes were determined essentially by the local pigment arrangement, but not by the higher-order structure, of the aggregate.