Femtosecond pulsed excitation of light-harvesting complexes creates oscillatory features in their response. This phenomenon has inspired a large body of work aimed at uncovering the origin of the coherent beatings and possible implications for function. Here we exploit site-directed mutagenesis to change the excitonic level structure in Fenna-Matthews-Olson (FMO) complexes and compare the coherences using broadband pump-probe spectroscopy. Our experiments detect two oscillation frequencies with dephasing on a picosecond timescale-both at 77 K and at room temperature. By studying these coherences with selective excitation pump-probe experiments, where pump excitation is in resonance only with the lowest excitonic state, we show that the key contributions to these oscillations stem from ground-state vibrational wavepackets. These experiments explicitly show that the coherences-although in the ground electronic state-can be probed at the absorption resonances of other bacteriochlorophyll molecules because of delocalization of the electronic excitation over several chromophores.

A structurally and compositionally well-defined and spectrally tunable artificial light-harvesting system has been constructed in which multiple organic dyes attached to a 3arm DNA nanostructure serve as an antenna conjugated to a photosynthetic reaction center isolated from Rhodobacter sphaeroides 2.4.1 (PDB 2J8C). The light energy absorbed by the dye molecules is transferred to the reaction center where charge separation takes place. The average number of DNA 3arm junctions per reaction center was tuned from 0.75 to 2.35. This DNAtemplated multi-chromophore system serves as a modular light-harvesting antenna that is capable of being optimized for its spectral properties, energy transfer efficiency and photostability, allowing one to adjust both the size and spectrum of the resulting structures. This may serve as a useful test-bed for developing nanostructured photonic systems. INTRODUCTION:

This review serves as an introduction to the variety of light-harvesting (LH) structures present in phototrophic prokaryotes. It provides an overview of the LH complexes of purple bacteria, green sulfur bacteria (GSB), acidobacteria, filamentous anoxygenic phototrophs (FAP), and cyanobacteria. Bacteria have adapted their LH systems for efficient operation under a multitude of different habitats and light qualities, performing both oxygenic (oxygen-evolving) and anoxygenic (non-oxygen-evolving) photosynthesis. For each LH system, emphasis is placed on the overall architecture of the pigment-protein complex, as well as any relevant information on energy transfer rates and pathways. This review addresses also some of the more recent findings in the field, such as the structure of the CsmA chlorosome baseplate and the whole-cell kinetics of energy transfer in GSB, while also pointing out some areas in need of further investigation.

Engineered cysteine residues near the primary electron donor (P) of the reaction center from the purple photosynthetic bacterium Rhodobacter sphaeroides were covalently conjugated to each of several dye molecules in order to explore the geometric design and spectral requirements for energy transfer between an artificial antenna system and the reaction center. An average of 2.5 fluorescent dye molecules were attached at specific locations near P. The enhanced absorbance cross-section afforded by conjugation of Alexa Fluor 660 dyes resulted in a 2.2-fold increase in the formation of reaction center charge-separated state upon intensity-limited excitation at 650 nm. The effective increase in absorbance cross-section resulting from the conjugation of two other dyes, Alexa Fluor 647 and Alexa Fluor 750, was also investigated. The key parameters that dictate the efficiency of dye-to-reaction center energy transfer and subsequent charge separation were examined using both steady-state and time-resolved fluorescence spectroscopy as well as transient absorbance spectroscopy techniques. An understanding of these parameters is an important first step toward developing more complex model light-harvesting systems integrated with reaction centers.

In this paper we report the steady-state optical properties of a series of site-directed mutants in the Fenna-Matthews-Olson (FMO) complex of Chlorobaculum tepidum, a photosynthetic green sulfur bacterium. The FMO antenna complex has historically been used as a model system for energy transfer due to the water-soluble nature of the protein, its stability at room temperature, as well as the availability of high-resolution structural data. Eight FMO mutants were constructed with changes in the environment of each of the bacteriochlorophyll a pigments found within each monomer of the homotrimeric FMO complex. Our results reveal multiple changes in low temperature absorption, as well as room temperature CD in each mutant compared to the wild-type FMO complex. These datasets were subsequently used to model the site energies of each pigment in the FMO complex by employing three different Hamiltonians from the literature. This enabled a basic approximation of the site energy shifts imparted on each pigment by the changed amino acid residue. These simulations suggest that, while the three Hamiltonians used in this work provide good fits to the wild-type FMO absorption spectrum, further efforts are required to obtain good fits to the mutant minus wild-type absorption difference spectra. This demonstrates that the use of FMO mutants can be a valuable tool to refine and iterate the current models of energy transfer in this system.

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Light-harvesting antenna complexes not only aid in the capture of solar energy for photosynthesis, but regulate the quantity of transferred energy as well. Light-harvesting regulation is important for protecting reaction center complexes from overexcitation, generation of reactive oxygen species, and metabolic overload. Usually, this regulation is controlled by the association of lightharvesting antennas with accessory quenchers such as carotenoids. One antenna complex, the Fenna-Matthews-Olson (FMO) antenna protein from green sulfur bacteria, completely lacks carotenoids and other known accessory quenchers. Nonetheless, the FMO protein is able to quench energy transfer in aerobic conditions effectively, indicating a previously unidentified type of regulatory mechanism. Through de novo sequencing MS, chemical modification, and mutagenesis, we have pinpointed the source of the quenching action to cysteine residues (Cys49 and Cys353) situated near two lowenergy bacteriochlorophylls in the FMO protein from Chlorobaculum tepidum. Removal of these cysteines (particularly removal of the completely conserved Cys353) through N-ethylmaleimide modification or mutagenesis to alanine abolishes the aerobic quenching effect. Electrochemical analysis and electron paramagnetic resonance spectra suggest that in aerobic conditions the cysteine thiols are converted to thiyl radicals which then are capable of quenching bacteriochlorophyll excited states through electron transfer photochemistry. This simple mechanism has implications for the design of bio-inspired light-harvesting antennas and the redesign of natural photosynthetic systems.photosynthesis | Fenna-Matthews-Olson protein | excitation quenching | thiyl radical | light-harvesting P hotosynthesis can be performed in the presence of oxygen (oxygenic photosynthesis, in which water is the electron source) or in its absence (anoxygenic photosynthesis, in which other reduced species such as sulfide are the electron sources) (1-3). Many bacteria performing anoxygenic photosynthesis contain type I reaction center (RC) protein complexes that use Fe-S clusters to transfer electrons to ferredoxin or related molecules after photoinduced charge separation (4-6). These Fe-S clusters are easily damaged by molecular oxygen. Therefore, anoxygenic phototrophs must use a pathway either to remove oxygen or to reduce the rate of photosynthesis whenever oxygen is encountered (1, 5).Phototrophic members of the bacterial phylum Chlorobi (green sulfur bacteria, GSBs) are anoxygenic phototrophs that contain such a type I RC. They also contain two peripheral antenna complexes that aid in regulating light absorption and energy transfer: the chlorosome and the homotrimeric Fenna-Matthews-Olson (FMO) protein complex. When oxygen is encountered, the bacteria are able to decrease the output of photosynthesis. This process involves creating trapping sites in the antenna complexes where de-excitation processes outcompete the rate of energy transfer to the RC, preventing RC damage. This process is well un...

Bacterial photosynthetic reaction centers (RCs) are promising materials for solar energy harvesting, due to their high quantum efficiency. A simple approach for making a photovoltaic device is to apply solubilized RCs and charge carrier mediators to the electrolyte of an electrochemical cell. However, the adsorption of analytes on the electrodes can affect the charge transfer from RCs to the electrodes. In this work, photovoltaic devices were fabricated incorporating RCs from purple bacteria, ubiquinone-10 (Q2), and cytochrome c (Cyt c) (the latter two species acting as redox mediators). The adsorption of each of these three species on the gold working electrode was investigated, and the roles of adsorbed species in the photocurrent generation and the cycle of charge transfer were studied by a series of photochronoamperometric, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and cyclic voltammetry (CV) tests. It was shown that both redox mediators were required for photocurrent generation; hence, the RC itself is likely unable to inject electrons into the gold electrode directly. The reverse redox reactions of mediators at the electrodes generates electrical current. Cyclic voltammograms for the RC-exposed gold electrode revealed a redox couple due to the adsorbed RC at ∼ +0.5 V (vs NHE), which confirmed that the RC was still redox active, upon adsorption to the gold. Photochronoamperometric studies also indicated that RCs adsorb, and are strongly bound to the surface of the gold, retaining functionality and contributing significantly to the process of photocurrent generation. Similar experiments showed the adsorption of Q2 and Cyt c on unmodified gold surfaces. It was indicated by the photochronoamperometric tests that the photocurrent derives from Q2-mediated charge transfer between the RCs and the gold electrode, while solubilized Cyt c mediates charge transfer between the P-side of adsorbed RC and the Pt counter electrode. Also, the stability of the adsorbed RCs and mediators was evaluated by measuring the photocurrent response over a period of 1 week. It is found that ∼46% of the adsorbed RCs remain active after a week under aerobic conditions. A significantly extended lifetime is expected by removing oxygen from the electrolyte and sealing the device.