Gregory S. Orf

Chlorosome antenna complexes from green photosynthetic bacteria

Chlorosomes are the distinguishing light-harvesting antenna complexes that are found in green photosynthetic bacteria. They contain bacteriochlorophyll (BChl) c, d, e in natural organisms, and recently through mutation, BChl f, as their principal light-harvesting pigments. In chlorosomes, these pigments self-assemble into large supramolecular structures that are enclosed inside a lipid monolayer to form an ellipsoid. The pigment assembly is dictated mostly by pigment–pigment interactions as opposed to protein–pigment interactions. On the bottom face of the chlorosome, the CsmA protein aggregates into a paracrystalline baseplate with BChl a, and serves as the interface to the next energy acceptor in the system. The exceptional light-harvesting ability at very low light conditions of chlorosomes has made them an attractive subject of study for both basic and applied science. This review, incorporating recent advancements, considers several important aspects of chlorosomes: pigment biosynthesis, organization of pigments and proteins, spectroscopic properties, and applications to bio-hybrid and bio-inspired devices.

Bacteriochlorophyll f: properties of chlorosomes containing the "forbidden chlorophyll"

The chlorosomes of green sulfur bacteria (GSB) are mainly assembled from one of three types of bacteriochlorophylls (BChls), BChls c, d, and e. By analogy to the relationship between BChl c and BChl d (20-desmethyl-BChl c), a fourth type of BChl, BChl f (20-desmethyl-BChl e), should exist but has not yet been observed in nature. The bchU gene (bacteriochlorophyllide C-20 methyltransferase) of the brown-colored green sulfur bacterium Chlorobaculum limnaeum was inactivated by conjugative transfer from Eshcerichia coli and homologous recombination of a suicide plasmid carrying a portion of the bchU. The resulting bchU mutant was greenish brown in color and synthesized BChl fF. The chlorosomes of the bchU mutant had similar size and polypeptide composition as those of the wild type (WT), but the Qy absorption band of the BChl f aggregates was blue-shifted 16 nm (705 nm vs. 721 nm for the WT). Fluorescence spectroscopy showed that energy transfer to the baseplate was much less efficient in chlorosomes containing BChl f than in WT chlorosomes containing BChl e. When cells were grown at high irradiance with tungsten or fluorescent light, the WT and bchU mutant had identical growth rates. However, the WT grew about 40% faster than the bchU mutant at low irradiance (10 μmol photons m−2 s-1). Less efficient energy transfer from BChl f aggregates to BChl a in the baseplate, the much slower growth of the strain producing BChl f relative to the WT, and competition from other phototrophs, may explain why BChl f is not observed naturally.

Dramatic Domain Rearrangements of the Cyanobacterial Orange Carotenoid Protein upon Photoactivation

Photosynthetic cyanobacteria make important contributions to global carbon and nitrogen budgets. A protein known as the orange carotenoid protein (OCP) protects the photosynthetic apparatus from damage by dissipating excess energy absorbed by the phycobilisome, the major light-harvesting complex in many cyanobacteria. OCP binds one carotenoid pigment, but the color of this pigment depends on conditions. It is orange in the dark and red when exposed to light. We modified the orange and red forms of OCP by using isotopically coded cross-linking agents and then analyzed the structural features by using liquid chromatography and tandem mass spectrometry. Unequivocal cross-linking pairs uniquely detected in red OCP indicate that, upon photoactivation, the OCP N-terminal domain (NTD) and C-terminal domain (CTD) reorient relative to each other. Our data also indicate that the intrinsically unstructured loop connecting the NTD and CTD not only is involved in the interaction between the two domains in orange OCP but also, together with the N-terminal extension, provides a structural buffer system facilitating an intramolecular breathing motion of the OCP, thus helping conversion back and forth from the orange to red form during the OCP photocycle. These results have important implications for understanding the molecular mechanism of action of cyanobacterial photoprotection.

Evidence for a cysteine-mediated mechanism of excitation energy regulation in a photosynthetic antenna complex

Significance All photosynthetic organisms face the challenge of absorbing solar energy and regulating its flow through their light-harvesting antennas across widely varying photic conditions. For anoxygenic phototrophs, this process is complicated by the need to downregulate photosynthetic output when oxygen is encountered. The Fenna–Matthews–Olson protein from green sulfur bacteria is able to quench excitations in aerobic conditions effectively despite its apparent lack of photoprotective accessory molecules, indicating a previously unidentified type of energy transfer regulation. In this study, we provide evidence for a novel energy-quenching mechanism involving cysteine–bacteriochlorophyll photochemistry. This interaction should be able to be programed into other natural or bio-inspired antennas, opening new possibilities for regulating these systems in response to excess light. 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 light-harvesting 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 low-energy 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.

Intensity Dependence of the Excited State Lifetimes and Triplet Conversion Yield in the Fenna–Matthews–Olson Antenna Protein

The Fenna-Matthews-Olson (FMO) protein is a soluble light-harvesting, bacteriochlorophyll a (BChl a) containing antenna complex found in green sulfur bacteria. We have measured time-resolved fluorescence and transient absorption at variable laser intensities at 298 and 77 K using FMO protein from Chlorobaculum tepidum prepared in both oxidizing and reducing environments. Fitting of the spectroscopic data shows that high laser intensities (i.e., above 10(13) photons × cm(-2) delivered per laser pulse) distort the intrinsic decay processes in this complex. At high laser intensities, both oxidized and reduced FMO samples behave similarly, exhibiting high levels of singlet-singlet annihilation. At lower laser intensities, the reduced protein mainly displays a singlet excited state lifetime of 2 ns, although upon oxidation, a 60 ps lifetime dominates. We also demonstrate that the apparent quantum yield of singlet-triplet intersystem crossing in the reduced FMO complex is ∼11% in the most favorable low laser intensities, with this yield decreasing and the probability of singlet-singlet annihilation yield increasing as laser intensity increases. After correcting for stimulated emission effects in the experiments, the actual maximum triplet yield is calculated to be ∼27%. Experiments at 77 K demonstrate that BChl a triplet states in FMO are localized on pigments no. 4 or 3, the lowest energy pigments in the complex. This study allows for a discussion of how BChl triplets form and evolve on the picosecond-to-nanosecond time scale, as well as whether triplet conversion is a physiologically relevant process.

Photophysical properties of the excited states of bacteriochlorophyll f in solvents and in chlorosomes.

Bacteriochlorophyll f (BChl f) is a photosynthetic pigment predicted nearly 40 years ago as a fourth potential member of the Chlorobium chlorophyll family (BChl c, d, and e). However, this pigment still has not been found in a naturally occurring organism. BChl c, d, and e are utilized by anoxygenic green photosynthetic bacteria for assembly of chlorosomes--large light-harvesting complexes that allow those organisms to survive in habitats with extremely low light intensities. Recently, using genetic methods on two different strains of Chlorobaculum limnaeum that naturally produce BChl e, two research groups produced mutants that synthesize BChl f and assemble it into chlorosomes. In this study, we present detailed investigations on spectral and dynamic characteristics of singlet excited and triplet states of BChl f with the application of ultrafast time-resolved absorption and fluorescence spectroscopies. The studies were performed on isolated BChl f in various solvents, at different temperatures, and on BChl f-containing chlorosomes in order to uncover any unusual or unfavorable properties that stand behind the lack of appearance of this pigment in natural environments.

Dynamics of Energy and Electron Transfer in the FMO-Reaction Center Core Complex from the Phototrophic Green Sulfur Bacterium Chlorobaculum tepidum

The reaction center core (RCC) complex and the RCC with associated Fenna-Matthews-Olson protein (FMO-RCC) complex from the green sulfur bacterium Chlorobaculum tepidum were studied comparatively by steady-state and time-resolved fluorescence (TRF) and femtosecond time-resolved transient absorption (TA) spectroscopies. The energy transfer efficiency from the FMO to the RCC complex was calculated to be ∼40% based on the steady-state fluorescence. TRF showed that most of the FMO complexes (66%), regardless of the fact that they were physically attached to the RCC, were not able to transfer excitation energy to the reaction center. The TA spectra of the RCC complex showed a 30-38 ps lifetime component regardless of the excitation wavelengths, which is attributed to charge separation. Excitonic equilibration was shown in TA spectra of the RCC complex when excited into the BChl a Qx band at 590 nm and the Chl a Qy band at 670 nm, while excitation at 840 nm directly populated the low-energy excited state and equilibration within the excitonic BChl a manifold was not observed. The TA spectra for the FMO-RCC complex excited into the BChl a Qx band could be interpreted by a combination of the excited FMO protein and RCC complex. The FMO-RCC complex showed an additional fast kinetic component compared with the FMO protein and the RCC complex, which may be due to FMO-to-RCC energy transfer.

Chemical activation of the cyanobacterial orange carotenoid protein

The effects of the Hofmeister series of ions on the activation of the orange carotenoid protein (OCP) from the inactive orange form to the active red form were tested. Kosmotropes led to lower OCP activation, whereas chaotropes led to greater OCP activation. Concentrations of thiocyanate exceeding 1.5 M dark activate the orange carotenoid protein to its red form. This chemically activated OCP was studied by UV–vis and circular dichroism spectroscopies. The chemically‐activated OCP quenches the fluorescence of phycobilisomes in vitro, to a level comparable to that of the light‐activated OCP.

Perturbation of bacteriochlorophyll molecules in Fenna-Matthews-Olson protein complexes through mutagenesis of cysteine residues.

The Fenna-Matthews-Olson (FMO) pigment-protein complex in green sulfur bacteria transfers excitation energy from the chlorosome antenna complex to the reaction center. In understanding energy transfer in the FMO protein, the individual contributions of the bacteriochlorophyll pigments to the FMO complexs absorption spectrum could provide detailed information with which molecular and energetic models can be constructed. The absorption properties of the pigments, however, are such that their spectra overlap significantly. To overcome this, we used site-directed mutagenesis to construct a series of mutant FMO complexes in the model green sulfur bacterium Chlorobaculum tepidum (formerly Chlorobium tepidum). Two cysteines at positions 49 and 353 in the C. tepidum FMO complex, which reside near hydrogen bonds between BChls 2 and 3, and their amino acid binding partner serine 73 and tyrosine 15, respectively, were changed to alanine residues. The resulting C49A, C353A, and C49A C353A double mutants were analyzed with a combination of optical absorption and circular dichroism (CD) spectroscopies. Our results revealed changes in the absorption properties of several underlying spectral components in the FMO complex, as well as the redox behavior of the complex in response to the reductant sodium dithionite. A high-resolution X-ray structure of the C49A C353A double mutant reveals that these spectral changes appear to be independent of any major structural rearrangements in the FMO mutants. Our findings provide important tests for theoretical calculations of the C. tepidum FMO absorption spectrum, and additionally highlight a possible role for cysteine residues in the redox activity of the pigment-protein complex.

The Fate of the Triplet Excitations in the Fenna–Matthews–Olson Complex

The fate of triplet excited states in the Fenna-Matthew-Olson (FMO) pigment-protein complex is studied by means of time-resolved nanosecond spectroscopy and exciton model simulations. Experiments reveal microsecond triplet excited-state energy transfer between the bacteriochlorophyll (BChl) pigments, but show no evidence of triplet energy transfer to molecular oxygen, which is known to produce highly reactive singlet oxygen and is the leading cause of photo damage in photosynthetic proteins. The FMO complex is exceptionally photo stable despite the fact it contains no carotenoids, which could effectively quench triplet excited states of (bacterio)chlorophylls and are usually found within pigment-protein complexes. It is inferred that the triplet excitation is transferred to the lowest energy pigment, BChl 3, within the FMO complex, whose triplet state energy is shifted by pigment-protein interactions below that of the singlet oxygen excitation. Thus, the energy transfer to molecular oxygen is blocked and the FMO does not need carotenoids for photo protection.