Timothy P. Causgrove

REDOX REGULATION OF ENERGY TRANSFER EFFICIENCY IN ANTENNAS OF GREEN PHOTOSYNTHETIC BACTERIA

The efficiency of energy transfer from the peripheral chlorosome antenna structure to the membrane‐bound antenna in green sulfur bacteria depends strongly on the redox potential of the medium. The fluorescence spectra and lifetimes indicate that efficient quenching pathways are induced in the chlorosome at high redox potential. The midpoint redox potential for the induction of this effect in isolated chlorosomes from Chlorobium vibrioforme is ‐146 mV at pH 7 (vs the normal hydrogen electrode), and the observed midpoint potential (n = 1) decreases by 60 mV per pH unit over the pH range7–10. Extraction of isolated chlorosomes with hexane has little effect on the redox‐induced quenching, indicating that the component(s) responsible for this effect are bound and not readily extractable. We have purified and partially characterized the trimeric water‐soluble bacteriochlorophyll a‐containing protein from the thermophilic green sulfur bacterium Chlorobium tepidum. This protein is located between the chlorosome and the membrane. Fluorescence spectra of the purified protein indicate that it also contains groups that quench excitations at high redox potential. The results indicate that the energy transfer pathway in green sulfur bacteria is regulated by redox potential. This regulation appears to operate in at least two distinct places in the energy transfer pathway, the oligomeric pigments in the interior of the chlorosome and in the bacteriochlorophyll a protein. The regulatory effect may serve to protect the cell against superoxide‐induced damage when oxygen is present. By quenching excitations before they reach the reaction center, reduction and subsequent autooxidation of the low potential electron acceptors found in these organisms is avoided.

Picosecond decay kinetics and quantum yield of fluorescence of the photoactive yellow protein from the halophilic purple phototrophic bacterium, Ectothiorhodospira halophila.

The photoactive yellow protein (PYP) has been previously shown to be partially bleached and red shifted (in less than 10 ns) by a pulse of laser excitation at the wavelength maximum (445 nm), to further bleach (k = 7.5 x 10(3) s(-1)), and then to slowly recover in the dark (k = 2.6 s(-1)) (Meyer, T. E., G. Tollin, J. H. Hazzard, and M. A. Cusanovich. 1989. Biophys. J. 56:559-564). The quantum yield for the formation of the fully bleached form was found to be 0.64. We have now shown that the yellow protein is weakly fluorescent with an emission maximum at 495 nm (which mirrors excitation at 445 nm) and a fluorescence quantum yield of 1.4 x 10(-3). Measurement of the picosecond kinetics of the fluorescence decay shows that approximately 90% of the emission occurs with a lifetime of 12 ps. This is in good agreement with the quantum yield determination, which suggests that a single quenching process (presumably the photochemical event) is primarily responsible for the excited state decay. The lifetime of the excited state of PYP is remarkably similar to that for the rise of the first photochemical intermediate of bacteriorhodopsin, and underscores the fundamental similarity in their photocycles despite a lack of structural relationship.

Energy transfer kinetics in whole cells and isolated chlorosomes of green photosynthetic bacteria

Time-resolved fluorescence spectroscopy and global data analysis techniques have been used to study the flow of excitations in antennae of the green photosynthetic bacteria Chloroflexus aurantiacus and Chlorobium vibrioforme f. thiosulfatophilum. The transfer of energy from bacteriochlorophyll (BChl) c in Chloroflexus or BChl d in Chlorobium to BChl a795 was resolved in both whole cells and isolated chlorosomes. In Chloroflexus, the decay of excitations in BChl c occurs in ∼16 ps and a corresponding rise in BChl a emission at 805 nm is detected in global analyses. This band then decays in 46 ps in whole cells due to energy transfer into the membrane. The 805 nm fluorescence in isolated chlorosomes shows a fast decay component similar to that of whole cells, which is consistent with trapping by residual membrane antenna complexes. In Chlorobium, the kinetics are sensitive to the presence of oxygen. Under anaerobic conditions, BChl d decays in 66 ps while the lifetime shortens to 11 ps in aerobic samples. The effect is reversible and occurs in both whole cells and isolated chlorosomes. Emission from BChl a is similarly affected by oxygen, indicating that oxidant-induced quenching can occur from all chlorosome pigments.

Förster energy transfer in chlorosomes of green photosynthetic bacteria.

Energy transfer properties of whole cells and chlorosome antenna complexes isolated from the green sulfur bacteria Chlorobium limicola (containing bacteriochlorophyll c), Chlorobium vibrioforme (containing bacteriochlorophyll d) and Pelodictyon phaeoclathratiforme (containing bacteriochlorophyll e) were measured. The spectral overlap of the major chlorosome pigment (bacteriochlorophyll c, d or, e) with the bacteriochlorophyll a B795 chlorosome baseplate pigment is greatest for bacteriochlorophyll c and smallest for bacteriochlorophyll e. The absorbance and fluorescence spectra of isolated chlorosomes were measured, fitted to gaussian curves and the overlap factors with B795 calculated. Energy transfer times from the bacteriochlorophyll c, d or e to B795 were measured in whole cells and the results interpreted in terms of the Förster theory of energy transfer.

Nanosecond temperature jump relaxation dynamics of cyclic β-hairpin peptides

The thermal unfolding of a series of 6-, 10-, and 14-mer cyclic β-hairpin peptides was studied to gain insight into the mechanism of formation of this important secondary structure. The thermodynamics of the transition were characterized using temperature dependent Fourier transform infrared spectroscopy. Thermodynamic data were analyzed using a two-state model which indicates increasing cooperativity along the series. The relaxation kinetics of the peptides in response to a laser induced temperature jump were probed using time-resolved infrared spectroscopy. Single exponential relaxation kinetics were observed and fit with a two-state model. The folding rate determined for these cyclic peptides is accelerated by some two orders of magnitude over the rate of a linear peptide that forms a β-hairpin. This observation supports the argument that the rate limiting step in the linear system is either stabilization of compact collapsed structures or rearrangement of collapsed structures over a barrier to achieve the native interstrand registry. Small activation energies for folding of these peptides obtained from an Arrhenius analysis of the rates imply a primarily entropic barrier, hence an organized transition state having specific stabilizing interactions.

Fluorescence lifetimes of dimers and higher oligomers of bacteriochlorophyll c from Chlorobium limicola

Fluorescence lifetimes have been measured for bacteriochlorophyll (BChl) c isolated from Chlorobium limicola in different states of aggregation in non-polar solvents. Two different homologs of BChl c were used, one with an isobutyl group at the 4 position, the other with n-propyl. Species previously identified as dimers (Olson and Pedersen 1990, Photosynth Res, this issue) decayed with lifetimes of 0.64 ns for the isobutyl homolog, 0.71 ns for n-propyl. Decay-associated spectra indicate that the absorption spectrum of the isobutyl dimer is slightly red-shifted from that of the n-propyl dimer. Aggregates absorbing maximally at 710 nm fluoresced with a principal lifetime of 3.1 ns, independent of the homolog used. In CCl4, only the isobutyl homolog forms a 747-nm absorbing oligomer spectrally similar to BChl c in vivo. This oligomer shows non-exponential fluorescence decay with lifetimes of 67 and 19 ps. Because the two components show different excitation spectra, the higher oligomer is probably a mixture of more than one species, both of which absorb at ∼747 nm.

Time‐resolved tryptophan fluorescence in photosynthetic reaction centers from Rhodobacter sphaeroides

Tryptophan fluorescence of reaction centers isolated from Rhodobacter sphaeroides, both stationary and time‐resolved, was studied. Fluorescence kinetics were found to fit best a sum of four discrete exponential components. Half of the initial amplitude was due to a component with a lifetime of ≅ 60 ps, belonging to Trp residues, capable of efficient transfer of excitation energy to bacteriochlorophyll molecules of the reaction center. The three other components seem to be emitted by Trp ground‐state conformers, unable to participate in such a transfer. Under the influence of intense actinic light, photooxidizing the reaction centers, the yield of stationary fluorescence diminished by ⋍1.5 times, while the number of the kinetic components and their life times remained practically unchanged. Possible implications of the observed effects for the primary photosynthesis events are considered.

Efficiency and Kinetics of Energy Transfer in Chlorosome Antennas from Green Photosynthetic Bacteria

Green photosynthetic bacteria are anoxygenic prokaryotes that contain a light gathering antenna complex known as a chlorosome (for reviews see references 1–2). The chlorosome is an ellipsoidal structure of dimensions approximately 100 nm × 30 nm × 12 nm in the green gliding bacterium Chloroflexus aurantiacus and somewhat larger in the green sulfur bacteria such as Chlorobium vibrioforme. The chlorosome is bounded by an envelope thought to be a lipid monolayer with some associated proteins. It is attached to the cytoplasmic side of the inner cell membrane, with additional membrane-bound antenna complexes and reaction centers embedded within the membrane (Fig. 1). Chlorosomes contain approximately 10,000 molecules of bacteriochlorophyll c, d or e and smaller amounts of BChl a and carotenoids.

Fast events in protein folding

The primary objective of this work was to develop a molecular understanding of how proteins achieve their native three-dimensional (folded) structures. This requires the identification and characterization of intermediates in the protein folding process on all relevant timescales, from picoseconds to seconds. The short timescale events in protein folding have been entirely unknown. Prior to this work, state-of-the-art experimental approaches were limited to milliseconds or longer, when much of the folding process is already over. The gap between theory and experiment is enormous: current theoretical and computational methods cannot realistically model folding processes with lifetimes longer than one nanosecond. This unique approach to employ laser pump-probe techniques that combine novel methods of laser flash photolysis with time-resolved vibrational spectroscopic probes of protein transients. In this scheme, a short (picosecond to nanosecond) laser photolysis pulse was used to produce an instantaneous pH or temperature jump, thereby initiating a protein folding or unfolding reaction. Structure-specific, time-resolved vibrational probes were then used to identify and characterize protein folding intermediates.