Light harvesting in purple bacteria does not rely on resonance fine-tuning in peripheral antenna complexes.

The ring-like peripheral light-harvesting complex 2 (LH2) expressed by many phototrophic purple bacteria is a popular model system in biological light-harvesting research due to its robustness, small size, and known crystal structure. Furthermore, the availability of structural variants with distinct electronic structures and optical properties has made this group of light harvesters an attractive testing ground for studies of structure-function relationships in biological systems. LH2 is one of several pigment-protein complexes for which a link between functionality and effects such as excitonic coherence and vibronic coupling has been proposed. While a direct connection has not yet been demonstrated, many such interactions are highly sensitive to resonance conditions, and a dependence of intra-complex dynamics on detailed electronic structure might be expected. To gauge the sensitivity of energy-level structure and relaxation dynamics to naturally occurring structural changes, we compare the photo-induced dynamics in two structurally distinct LH2 variants. Using polarization-controlled 2D electronic spectroscopy at cryogenic temperatures, we directly access information on dynamic and static disorder in the complexes. The simultaneous optimal spectral and temporal resolution of these experiments further allows us to characterize the ultrafast energy relaxation, including exciton transport within the complexes. Despite the variations in PPC molecular structure manifesting as clear differences in electronic structure and disorder, the energy-transport and-relaxation dynamics remain remarkably similar. This indicates that the light-harvesting functionality of purple bacteria within a single LH2 complex is highly robust to structural perturbations and likely does not rely on finely tuned electronic- or electron-vibrational resonance conditions.

Enhancement of the Photocurrent of a Single Photosystem I Complex by the Localized Plasmon of a Gold Nanorod.

A combination of conductive atomic force microscopy (AFM) and confocal fluorescence microscopy was used to measure photocurrents passing through single trimeric photosytem I (PSI) complexes located in the vicinity of single gold nanorods (AuNRs). Simultaneous excitation of PSI and of the AuNR longitudinal plasmon mode and detection of photocurrents from individual PSI in relation to the position of single AuNRs enable insight into plasmon-induced phenomena that are otherwise inaccessible in ensemble experiments. We have observed photocurrent enhancement by the localized plasmons by a factor of 2.9 on average, with maximum enhancement values of up to 8. Selective excitation of the longitudinal plasmon modes by the polarization of the excitation laser enables controllable switch-on of the photocurrent enhancement. The dependence of the extent of enhancement on the distance between PSI and AuNRs indicates that, apart from the enhancement of absorption, there is an additional enhancement mechanism affecting directly the electron transport process. The present study provides deeper insight into the molecular mechanisms of plasmon-enhanced photocurrents, not only in PSI but also potentially in other systems as well.

Two-photon absorption and excitation spectroscopy of carotenoids, chlorophylls and pigment-protein complexes.

In addition to (bacterio)chlorophylls, (B)Chls, photosynthetic pigment-protein complexes bind carotenoids (Cars) that fulfil various important functions which are not fully understood, yet. However, certain excited states of Cars are optically one-photon forbidden ("dark") and can potentially undergo excitation energy transfer (EET) to (B)Chls following two-photon absorption (TPA). The amount of EET is reflected by the differences in TPA and two-photon excitation (TPE) spectra of a complex (multi-pigment) system. Since it is technically and analytically demanding to resolve optically forbidden states, different studies reported varying contributions of Cars and Chls to TPE/TPA spectra. In a study using well-defined 1 : 1 Car-tetrapyrrole dyads TPE contributions of tetrapyrrole molecules, including Chls, and Cars were measured. In these experiments, TPE of Cars dominated over Chl a TPE in a broad wavelength range. Another study suggested only minor contributions of Cars to TPE spectra of pigment-protein complexes such as the plant main light-harvesting complex (LHCII), in particular for wavelengths longer than ∼600/1200 nm. By joining forces and a combined analysis of all available data by both teams, we try to resolve this apparent contradiction. Here, we demonstrate that reconstruction of a wide spectral range of TPE for LHCII and photosystem I (PSI) requires both, significant Car and Chl contributions. Direct comparison of TPE spectra obtained in both studies demonstrates a good agreement of the primary data. We conclude that in TPE spectra of LHCII and PSI, the contribution of Chls is dominating above 600/1200 nm, whereas the contributions of forbidden Car states increase particularly at wavelengths shorter than 600/1200 nm. Estimates of Car contributions to TPA as well as TPE spectra are given for various wavelengths.

Photosynthetic Light-Harvesting (Antenna) Complexes-Structures and Functions.

Chlorophylls and bacteriochlorophylls, together with carotenoids, serve, noncovalently bound to specific apoproteins, as principal light-harvesting and energy-transforming pigments in photosynthetic organisms. In recent years, enormous progress has been achieved in the elucidation of structures and functions of light-harvesting (antenna) complexes, photosynthetic reaction centers and even entire photosystems. It is becoming increasingly clear that light-harvesting complexes not only serve to enlarge the absorption cross sections of the respective reaction centers but are vitally important in short- and long-term adaptation of the photosynthetic apparatus and regulation of the energy-transforming processes in response to external and internal conditions. Thus, the wide variety of structural diversity in photosynthetic antenna "designs" becomes conceivable. It is, however, common for LHCs to form trimeric (or multiples thereof) structures. We propose a simple, tentative explanation of the trimer issue, based on the 2D world created by photosynthetic membrane systems.

Silver Island Film for Enhancing Light Harvesting in Natural Photosynthetic Proteins.

The effects of combining naturally evolved photosynthetic pigment-protein complexes with inorganic functional materials, especially plasmonically active metallic nanostructures, have been a widely studied topic in the last few decades. Besides other applications, it seems to be reasonable using such hybrid systems for designing future biomimetic solar cells. In this paper, we describe selected results that point out to various aspects of the interactions between photosynthetic complexes and plasmonic excitations in Silver Island Films (SIFs). In addition to simple light-harvesting complexes, like peridinin-chlorophyll-protein (PCP) or the Fenna-Matthews-Olson (FMO) complex, we also discuss the properties of large, photosynthetic reaction centers (RCs) and Photosystem I (PSI)-both prokaryotic PSI core complexes and eukaryotic PSI supercomplexes with attached antenna clusters (PSI-LHCI)-deposited on SIF substrates.

Insights into the binding behavior of native and non-native cytochromes to photosystem I from Thermosynechococcus elongatus.

The binding of photosystem I (PS I) from Thermosynechococcus elongatus to the native cytochrome (cyt) c6 and cyt c from horse heart (cyt cHH) was analyzed by oxygen consumption measurements, isothermal titration calorimetry (ITC), and rigid body docking combined with electrostatic computations of binding energies. Although PS I has a higher affinity for cyt cHH than for cyt c6, the influence of ionic strength and pH on binding is different in the two cases. ITC and theoretical computations revealed the existence of unspecific binding sites for cyt cHH besides one specific binding site close to P700 Binding to PS I was found to be the same for reduced and oxidized cyt cHH Based on this information, suitable conditions for cocrystallization of cyt cHH with PS I were found, resulting in crystals with a PS I:cyt cHH ratio of 1:1. A crystal structure at 3.4-Å resolution was obtained, but cyt cHH cannot be identified in the electron density map because of unspecific binding sites and/or high flexibility at the specific binding site. Modeling the binding of cyt c6 to PS I revealed a specific binding site where the distance and orientation of cyt c6 relative to P700 are comparable with cyt c2 from purple bacteria relative to P870 This work provides new insights into the binding modes of different cytochromes to PS I, thus facilitating steps toward solving the PS I-cyt c costructure and a more detailed understanding of natural electron transport processes.

The origin of the "dark" absorption band near 675 nm in the purple bacterial core light-harvesting complex LH1: two-photon measurements of LH1 and its subunit B820.

A comparative two-photon excitation spectroscopic study of the exciton structure of the core antenna complex (LH1) and its subunit B820 was carried out. LH1 and its subunit B820 were isolated from cells of the carotenoid-less mutant G9 of Rhodospirillum rubrum. The measurements were performed by two-photon pump-probe spectroscopy. Samples were excited by 70 fs pulses at 1390 nm at a frequency of 1 kHz. Photoinduced absorption changes were recorded in the spectral range from 780 to 1020 nm for time delays of the probe pulse relative to the pump pulse in the - 1.5 to 11 ps range. All measurements were performed at room temperature. Two-photon excitation caused bleaching of exciton bands (k = 0, k = ± 1) of the circular bacteriochlorophyll aggregate of LH1. In the case of the B820 subunit, two-photon excitation did not cause absorption changes in this spectral range. It is proposed that in LH1 upper exciton branch states are mixed with charge-transfer (CT) states. In B820 such mixing is absent, precluding two-photon excitation in this spectral region. Usually, CT states are optically "dark", i.e., one photon-excitation forbidden. Thus, their investigation is rather complicated by conventional spectroscopic methods. Thus, our study provides a novel approach to investigate CT states and their interaction(s) with other excited states in photosynthetic light-harvesting complexes and other molecular aggregates.

Two-photon excitation spectroscopy of photosynthetic light-harvesting complexes and pigments.

In addition to (bacterio)chlorophylls, (B)Chls, light-harvesting complexes (LHCs) bind carotenoids, and/or their oxygen derivatives, xanthophylls. Xanthophylls/carotenoids have pivotal functions in LHCs: in stabilization of the structure, as accessory light-harvesting pigments and, probably most importantly, in photoprotection. Xanthophylls are assumed to be involved in the not yet fully understood mechanism of energy-dependent (qE) non-photochemical quenching of Chl fluorescence (NPQ) in higher plants and algae. The so called "xanthophyll cycle" appears to be crucial in this regard. The molecular mechanism(s) of xanthophyll involvement in qE/NPQ have not been established, yet. Moreover, excitation energy transfer (EET) processes involving carotenoids are also difficult to study, due to the fact that transitions between the ground state (S0, 11Ag-) and the lowest excited singlet state (S1, 21Ag-) of carotenoids are optically one-photon forbidden ("dark"). Two-photon excitation spectroscopic techniques have been used for more than two decades to study one-photon forbidden states of carotenoids. In the current study, two-photon excitation profiles of LHCII samples containing different xanthophyll complements were measured in the presumed 11Ag- → 21Ag- (S0 → S1) transition spectral region of the xanthophylls, as well as for isolated chlorophylls a and b in solution. The results indicate that direct two-photon excitation of Chls in this spectral region is dominant over that by xanthophylls. Implications of the results for proposed mechanism(s) of qE/NPQ will be discussed.

Spectrally selective fluorescence imaging of Chlorobaculum tepidum reaction centers conjugated to chelator-modified silver nanowires.

A polyhistidine tag (His-tag) present on Chlorobaculum tepidum reaction centers (RCs) was used to immobilize photosynthetic complexes on a silver nanowire (AgNW) modified with nickel-chelating nitrilo-triacetic acid (Ni-NTA). The optical properties of conjugated nanostructures were studied using wide-field and confocal fluorescence microscopy. Plasmonic enhancement of RCs conjugated to AgNWs was observed as their fluorescence intensity dependence on the excitation wavelength does not follow the excitation spectrum of RC complexes in solution. The strongest effect of plasmonic interactions on the emission intensity of RCs coincides with the absorption spectrum of AgNWs and is observed for excitation into the carotenoid absorption. From the absence of fluorescence decay shortening, we attribute the emission enhancement to increase of absorption in RC complexes.

Carotenoid-to-bacteriochlorophyll energy transfer through vibronic coupling in LH2 from Phaeosprillum molischianum.

The peripheral light-harvesting antenna complex (LH2) of purple photosynthetic bacteria is an ideal testing ground for models of structure-function relationships due to its well-determined molecular structure and ultrafast energy deactivation. It has been the target for numerous studies in both theory and ultrafast spectroscopy; nevertheless, certain aspects of the convoluted relaxation network of LH2 lack a satisfactory explanation by conventional theories. For example, the initial carotenoid-to-bacteriochlorophyll energy transfer step necessary on visible light excitation was long considered to follow the Förster mechanism, even though transfer times as short as 40 femtoseconds (fs) have been observed. Such transfer times are hard to accommodate by Förster theory, as the moderate coupling strengths found in LH2 suggest much slower transfer within this framework. In this study, we investigate LH2 from Phaeospirillum (Ph.) molischianum in two types of transient absorption experiments-with narrowband pump and white-light probe resulting in 100 fs time resolution, and with degenerate broadband 10 fs pump and probe pulses. With regard to the split Qx band in this system, we show that vibronically mediated transfer explains both the ultrafast carotenoid-to-B850 transfer, and the almost complete lack of transfer to B800. These results are beyond Förster theory, which predicts an almost equal partition between the two channels.

Bioelectronic Circuit on a 3D Electrode Architecture: Enzymatic Catalysis Interconnected with Photosystem I.

Artificial light-driven signal chains are particularly important for the development of systems converting light into a current, into chemicals or for light-induced sensing. Here, we report on the construction of an all-protein, light-triggered, catalytic circuit based on photosystem I, cytochrome c (cyt c) and human sulfite oxidase (hSOX). The defined assembly of all components using a modular design results in an artificial biohybrid electrode architecture, combining the photophysical features of PSI with the biocatalytic properties of hSOX for advanced light-controlled bioelectronics. The working principle is based on a competitive switch between electron supply from the electrode or by enzymatic substrate conversion.

Solution structure of monomeric and trimeric photosystem I of Thermosynechococcus elongatus investigated by small-angle X-ray scattering.

The structure of monomeric and trimeric photosystem I (PS I) of Thermosynechococcus elongatus BP1 (T. elongatus) was investigated by small-angle X-ray scattering (SAXS). The scattering data reveal that the protein-detergent complexes possess radii of gyration of 58 and 78 Å in the cases of monomeric and trimeric PS I, respectively. The results also show that the samples are monodisperse, virtually free of aggregation, and contain empty detergent micelles. The shape of the protein-detergent complexes can be well approximated by elliptical cylinders with a height of 78 Å. Monomeric PS I in buffer solution exhibits minor and major radii of the elliptical cylinder of about 50 and 85 Å, respectively. In the case of trimeric PS I, both radii are equal to about 110 Å. The latter model can be shown to accommodate three elliptical cylinders equal to those describing monomeric PS I. A structure reconstitution also reveals that the protein-detergent complexes are larger than their respective crystal structures. The reconstituted structures are larger by about 20 Å mainly in the region of the hydrophobic surfaces of the monomeric and trimeric PS I complexes. This seeming contradiction can be resolved by the addition of a detergent belt constituted by a monolayer of dodecyl-ß-D-maltoside molecules. Assuming a closest possible packing, a number of roughly 1024 and 1472 detergent molecules can be determined for monomeric and trimeric PS I, respectively. Taking the monolayer of detergent molecules into account, the solution structure can be almost perfectly modeled by the crystal structures of monomeric and trimeric PS I.

The small regulatory RNA SyR1/PsrR1 controls photosynthetic functions in cyanobacteria.

Little is known so far about RNA regulators of photosynthesis in plants, algae, or cyanobacteria. The small RNA PsrR1 (formerly SyR1) has been discovered in Synechocystis sp PCC 6803 and appears to be widely conserved within the cyanobacterial phylum. Expression of PsrR1 is induced shortly after a shift from moderate to high-light conditions. Artificial overexpression of PsrR1 led to a bleaching phenotype under moderate light growth conditions. Advanced computational target prediction suggested that several photosynthesis-related mRNAs could be controlled by PsrR1, a finding supported by the results of transcriptome profiling experiments upon pulsed overexpression of this small RNA in Synechocystis sp PCC 6803. We confirmed the interaction between PsrR1 and the ribosome binding regions of the psaL, psaJ, chlN, and cpcA mRNAs by mutational analysis in a heterologous reporter system. Focusing on psaL as a specific target, we show that the psaL mRNA is processed by RNase E only in the presence of PsrR1. Furthermore, we provide evidence for a posttranscriptional regulation of psaL by PsrR1 in the wild type at various environmental conditions and analyzed the consequences of PsrR1-based regulation on photosystem I. In summary, computational and experimental data consistently establish the small RNA PsrR1 as a regulatory factor controlling photosynthetic functions.

Silver island film substrates for ultrasensitive fluorescence detection of (bio)molecules.

A silver island film (SIF) substrate was used to demonstrate that Metal-Enhanced Fluorescence (MEF) is a powerful tool to enable detection of emission from (bio)molecules at very low concentrations. The experiments were carried out with the Fenna-Matthews-Olson (FMO) pigment-protein complex from the photosynthetic green sulfur bacterium Chlorobaculum tepidum. FMO was diluted to a level, at which no emission was detectable on a glass substrate. In contrast, the fluorescence of FMO was readily observed on the SIF substrate, even though the emission wavelength of FMO is displaced by over 300 nm from the maximum of the plasmon resonance of the SIF layer. Estimated enhancements of the fluorescence intensity of FMO on SIF are about 40-fold. The enhancement factor correlates with the improvement of the signal-to-noise ratio for FMO emission on SIF substrates.

Evidence for a role of VIPP1 in the structural organization of the photosynthetic apparatus in Chlamydomonas.

The vesicle-inducing protein in plastids (VIPP1) was suggested to play a role in thylakoid membrane formation via membrane vesicles. As this functional assignment is under debate, we investigated the function of VIPP1 in Chlamydomonas reinhardtii. Using immunofluorescence, we localized VIPP1 to distinct spots within the chloroplast. In VIPP1-RNA interference/artificial microRNA cells, we consistently observed aberrant, prolamellar body-like structures at the origin of multiple thylakoid membrane layers, which appear to coincide with the immunofluorescent VIPP1 spots and suggest a defect in thylakoid membrane biogenesis. Accordingly, using quantitative shotgun proteomics, we found that unstressed vipp1 mutant cells accumulate 14 to 20% less photosystems, cytochrome b(6)f complex, and ATP synthase but 30% more light-harvesting complex II than control cells, while complex assembly, thylakoid membrane ultrastructure, and bulk lipid composition appeared unaltered. Photosystems in vipp1 mutants are sensitive to high light, which coincides with a lowered midpoint potential of the Q(A)/Q(A)(-) redox couple and increased thermosensitivity of photosystem II (PSII), suggesting structural defects in PSII. Moreover, swollen thylakoids, despite reduced membrane energization, in vipp1 mutants grown on ammonium suggest defects in the supermolecular organization of thylakoid membrane complexes. Overall, our data suggest a role of VIPP1 in the biogenesis/assembly of thylakoid membrane core complexes, most likely by supplying structural lipids.

Unidirectional Photocurrent of Photosystem I on π-System-Modified Graphene Electrodes: Nanobionic Approaches for the Construction of Photobiohybrid Systems.

One major vital element of the oxygenic photosynthesis is photosystem I (PSI). We report on the construction of graphene-based nanohybrid light-harvesting architectures consisting of PSI supercomplexes adsorbed onto π-system-modified graphene interfaces. The light-driven nanophotobioelectrochemical architectures have been designed on a modified carbon surface, on the basis of π-π-stacking interactions between polycyclic aromatic compounds and graphene. As a result of the remarkable features of graphene and the feasibility of purposeful surface property adjustment, well-defined photoelectrochemical responses have been displayed by the nanophotohybrid electrodes. In particular, the PSI-graphene electrodes utilizing naphthalene derivatives provided a suitable surface for the adsorption of PSI and display already at the open circuit potential (OCP) a high cathodic photocurrent output of 4.5 ± 0.1 µA/cm(2). By applying an overpotential and addition of a soluble electron acceptor (methyl viologen), the photocurrent density can be further magnified to 20 ± 0.5 µA/cm(2). On the contrary, the investigated anthracene-based PSI-graphene electrodes exhibit considerably smaller and not very directed photoelectrochemical responses. This study grants insights into the influences of different polycyclic aromatic compounds acting as an interface between the very large protein supercomplex PSI and graphene while supporting the electrochemical communication of the biomolecule with the electrode. It needs to be emphasized that solely the naphthalene-based photoelectrodes reveal unidirectional cathodic photocurrents, establishing the feasibility of utilizing this advanced approach for the construction of next-generation photovoltaic devices.

Chlorophyll a phytylation is required for the stability of photosystems I and II in the cyanobacterium Synechocystis sp. PCC 6803.

In oxygenic phototrophic organisms, the phytyl 'tail' of chlorophyll a is formed from a geranylgeranyl residue by the enzyme geranylgeranyl reductase. Additionally, in oxygenic phototrophs, phytyl residues are the tail moieties of tocopherols and phylloquinone. A mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking geranylgeranyl reductase, ΔchlP, was compared to strains with specific deficiencies in either tocopherols or phylloquinone to assess the role of chlorophyll a phytylatation (versus geranylgeranylation). The tocopherol-less Δhpt strain grows indistinguishably from the wild-type under 'standard' light photoautotrophic conditions, and exhibited only a slightly enhanced rate of photosystem I degradation under strong irradiation. The phylloquinone-less ΔmenA mutant also grows photoautotrophically, albeit rather slowly and only at low light intensities. Under strong irradiation, ΔmenA retained its chlorophyll content, indicative of stable photosystems. ΔchlP may only be cultured photomixotrophically (due to the instability of both photosystems I and II). The increased accumulation of myxoxanthophyll in ΔchlP cells indicates photo-oxidative stress even under moderate illumination. Under high-light conditions, ΔchlP exhibited rapid degradation of photosystems I and II. In conclusion, the results demonstrate that chlorophyll a phytylation is important for the (photo)stability of photosystems I and II, which, in turn, is necessary for photoautotrophic growth and tolerance of high light in an oxygenic environment.

Advanced unidirectional photocurrent generation via cytochrome c as reaction partner for directed assembly of photosystem I.

Conversion of light into an electrical current based on biohybrid systems mimicking natural photosynthesis is becoming increasingly popular. Photosystem I (PSI) is particularly useful in such photo-bioelectrochemical devices. Herein, we report on a novel biomimetic approach for an effective assembly of photosystem I with the electron transfer carrier cytochrome c (cyt c), deposited on a thiol-modified gold-surface. Atomic force microscopy and surface plasmon resonance measurements have been used for characterization of the assembly process. Photoelectrochemical experiments demonstrate a cyt c mediated generation of an enhanced unidirectional cathodic photocurrent. Here, cyt c can act as a template for the assembly of an oriented and dense layer of PSI and as wiring agent to direct the electrons from the electrode towards the photosynthetic reaction center of PSI. Furthermore, three-dimensional protein architectures have been formed via the layer-by-layer deposition technique resulting in a successive increase in photocurrent densities. An intermittent cyt c layer is essential for an efficient connection of PSI layers with the electrode and for an improvement of photocurrent densities.

Correction to "Excitation Energy Transfer between Higher Excited States of Photosynthetic Pigments: 1. Carotenoids Intercept and Remove B Band Excitations".

[This corrects the article DOI: 10.1021/acsomega.3c05895.].