Vibrational equilibration in absorption difference spectra of chlorophyll a.

We describe Franck-Condon simulations of vibrational cooling effects on absorption difference spectra in chlorophyll a (Chl a). The relative contributions of vibrational equilibration in the electronic ground and excited states depend on the pump and probe wavelengths. For Franck-Condon-active vibrational modes exhibiting small Huang-Rhys factors (S < 0.1, characteristic in Chl a pigments), vibrational thermalization causes essentially no spectral changes when the origin band is excited. Significant spectral evolution does occur for S < 0.1 when the 0-1 and 1.0 (hot) vibronic bands are excited. However, vibrational equilibration in these cases causes no spectral shifting in the empirical photobleaching/stimulated emission band maximum. This result bears on the interpretation of time-resolved absorption difference spectra of Chl a-containing antennae such as the Chl a/b light-harvesting peripheral antenna of photosystem II.     

Optical absorption spectra of deoxy- and oxyhemoglobin in the temperature range 300-20 K. Relation with protein dynamics

We have studied the optical absorption spectra of human deoxy- and oxyhemoglobin in the temperature range 300-20 K and in the wavelength range 350-1350 nm. By lowering the temperature, a narrowing and a shift of all bands were observed together with a sizeable increase of the integrated intensities of the charge-transfer bands of deoxyhemoglobin. At all temperatures the spectra are in full agreement with the band assignment previously suggested in the literature and no new relevant bands have been detected for both deoxy- and oxyhemoglobin. Analysis of the first and second moment of the bands, within the framework of the harmonic Franck-Condon approximation, gave information on the dynamic properties of the heme in the heme pocket.     

An inexpensive device for recording difference absorption spectra at low temperature (-196 degrees)

An inexpensive and very simple device is described for low-temperature difference absorption spectra of biological preparations. The spectra recorded from 380 to 650 nm have a sufficient resolution without “noise”.     

Hydrogen-deuterium exchange mass spectrometry reveals the interaction of Fenna-Matthews-Olson protein and chlorosome CsmA protein

In green-sulfur bacterial photosynthesis, excitation energy absorbed by a peripheral antenna structure known as the chlorosome is sequentially transferred through a baseplate protein to the Fenna-Matthews-Olson (FMO) antenna protein and into the reaction center, which is embedded in the cytoplasmic membrane. The molecular details of the optimized photosystem architecture required for efficient energy transfer are only partially understood. We address here the question of how the baseplate interacts with the FMO protein by applying hydrogen/deuterium exchange coupled with enzymatic digestion and mass spectrometry analysis to reveal the binding interface of the FMO antenna protein and the CsmA baseplate protein. Several regions on the FMO protein, represented by peptides consisting of 123-129, 140-149, 150-162, 191-208, and 224-232, show significant decreases of deuterium uptake after CsmA binding. The results indicate that the CsmA protein interacts with the Bchl a #1 side of the FMO protein. A global picture including peptide-level details for the architecture of the photosystem from green-sulfur bacteria can now be drawn.     

Deriving the intermediate spectra and photocycle kinetics from time-resolved difference spectra of bacteriorhodopsin. The simpler case of the recombinant D96N protein.

The bacteriorhodopsin photocycle contains more than five spectrally distinct intermediates, and the complexity of their interconversions has precluded a rigorous solution of the kinetics. A representation of the photocycle of mutated D96N bacteriorhodopsin near neutral pH was given earlier (Váró, G., and J. K. Lanyi. 1991. Biochemistry. 30:5008-5015) as BRhv-->K<==>L<==>M1-->M2--> BR. Here we have reduced a set of time-resolved difference spectra for this simpler system to three base spectra, each assumed to consist of an unknown mixture of the pure K, L, and M difference spectra represented by a 3 x 3 matrix of concentration values between 0 and 1. After generating all allowed sets of spectra for K, L, and M (i.e., M1 + M2) at a 1:50 resolution of the matrix elements, invalid solutions were eliminated progressively in a search based on what is expected, empirically and from the theory of polyene excited states, for rhodopsin spectra. Significantly, the average matrix values changed little after the first and simplest of the search criteria that disallowed negative absorptions and more than one maximum for the M intermediate. We conclude from the statistics that during the search the solutions strongly converged into a narrow region of the multidimensional space of the concentration matrix. The data at three temperatures between 5 and 25 degrees C yielded a single set of spectra for K, L, and M; their fits are consistent with the earlier derived photocycle model for the D96N protein.     

Electronic excited states and excitation transfer kinetics in the Fenna-Matthews-Olson protein of the photosynthetic bacterium Prosthecochloris aestuarii at low temperatures

The molecular structure-function relationship of the Fenna-Matthews-Olson light-harvesting complex of the photosynthetic green bacterium Prosthecochloris aestuarii has been investigated. It has been assumed that the electronic excited states responsible for the function (transfer of electronic excitation energy) result from the dipole-dipole interactions between the bacteriochlorophyll molecules bound to the polypeptide chain of the complex at a specific three-dimensional geometry. The molecular structure-electronic excited states relationship has been addressed on the basis of simultaneous simulations of several spectroscopic observations. Current electronic excited state models for the Fenna-Matthews-Olson complex have generally been based on obtaining an optimal match between the information contents of the optical steady-state spectra and the bacteriochlorophyll organization. Recent kinetic and spectral information gathered from ultrafast time-resolved measurements have not yet been used effectively for further refinement of the excited state models and for quantification of the relation between the excited states and the energy transfer processes. In this study, we have searched for a model that not only can explain the key features of several steady-state spectra but also the temporal and spectral evolution observed in a recent absorption difference experiment and we have discussed the implications of this model for equilibration of the electronic excitation energy in systems at low temperatures. Received: 12 June 1998 / Revised version: 19 October 1998 / Accepted: 30 November 1998     

Computational study of the absorption spectra of green fluorescent protein mutants

In this work, we present a theoretical study of the relationship between molecular structure and the red-shift in absorption spectra of S65G and S65T green fluorescent protein (GFP) mutants. To identify the effects of the protein environment, we combined results from molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics calculations to obtain structural properties, and applied time-dependent density functional theory to calculate the excitation energies. By using results from the MD simulations, we were able to provide a systematic analysis of the structural details that may effect the red-shift in the absorption spectra when taking into account temperature effects. Furthermore, a detailed study of hydrogen bonding during the MD simulations demonstrated differences between S65G and S65T, for example, regarding hydrogen bonding with Glu222. An analysis of the absorption spectra for different forms of the chromophore emphasized the dominance of the anionic forms in solution for the S65G and S65T GFP mutants.     

Phase fluorimetric lifetime spectra. I. In algal cells at 77 K

A new method for decomposing fluorescence emission spectra into their elementary components, based on the simultaneous recording of fluorescence intensity and lifetime vs. the emission wavelength, has been applied to the spectra of algal cells at liquid nitrogen temperature. A model of Gaussian components fits both τ(λ) and F(λ) spectra with the same parameters. The fluorescence lifetimes have been measured by phase fluorimetry at two modulation frequencies: 29 and 139 MHz. The final Gaussian decomposition is able to describe both the 29 and 139 MHz spectra. The following conclusions concerning the fluorescence spectra of Chlorella cells at 77 K can be drawn. These conclusions are also valid with minor changes for the other examined species. (1) An overlapping of different emitting bands occurs in all the spectra; therefore, a direct lifetime reading from phase delay measurement necessitates measurements being made at several frequencies. (2) At the F_max fluorescence level, the lifetime values of the two emissions usually associated with variable fluorescence are 0.53 ns (for B′₁; λ peak 688 nm), and 1.46 ns (for B′₂; λ peak 698 nm); these lifetimes are shorter than those we have measured at room temperature (approx. 1.8 ns). (3) Superimposed on B′₁ and B′₂ and with approximatively the same peak location, two long-lifetime components (B″₁, 4.8 ns; B″₂, 5.6 ns) are present. Two hypotheses can be proposed to explain these emissions: (i) the long-lifetime components arise from subsets of chlorophyll a disconnected from the functional antenna by the cooling process; and (ii) charge recombination in reaction centers leads to delayed fluorescence. (4) In the λ > 710 nm region, two main bands are required to describe the so-called Photosystem I emission: B₃ (0.8 ns; λ peak 715 nm) and B₄ (3.3 ns; λ peak 724 nm). The former band, usually unresolved in the amplitude fluorescence spectra, is a specific finding from lifetime measurements and has been associated with the antenna core of Photosystem I. No additional information has been obtained for B₄. A supplementary small band (B₅, 0.40 ns; $\text{λ}\text{peak}\text{? 740}\text{nm}$) is necessary to take into account the frequency effect and the τ(λ) decrease in the λ > 740 nm spectral range.     

Chlorophylls attached to lipid and protein globules, absorption and fluorescence spectra, photo-oxidation.

The precipitation of chlorophylls upon lipid and protein globules suspended in an aqueous buffer yields a partial model of photosynthetic membranes. The absorption and fluorescence spectra of the model are investigated as well as the photo-oxidation of the chlorophylls (bleaching) by dissolved oxygen. It is shown that pigment--pigment interactions occur in such systems, by (a) the appearance of absorption bands characteristic of crystalline or highly ordered chlorophyll at high pigment concentrations, (b) the chlorophyll a-type of fluorescence of systems containing chlorophyll a and chlorophyll b where the latter is selectively excited, and (c) the kinetics of photo-oxidation which suggest that chlorophylls can only be bleached when they are dimerized.     

Thermal perturbation difference spectra and D2O vs H2O isothermal difference spectra of protein-model aromatic chromophores.

The thermal perturbation difference spectra of phenolic and indolic chromophores in water resemble the isothermal D₂O and H₂O spectra of these chromophores. For phenols approximately equal Δ? values are obtained in both types of spectra, but for their methyl ethers Δ? values of D₂O vs H₂O spectra are about half of those of the thermal perturbation spectra. Phenols and their methyl ethers were studied in deuterated ethylene glycol and glycerol vs the corresponding protiated solvent, and in nonprotic solvents containing 0.25–4% D₂O or H₂O. For phenols in D₂O vs H₂O, about one-third to one-half of the difference spectrum is attributed to solvent structure difference, and the remainder to the effects of replacing OH by OD and to differences in accepting hydrogen bonds from D₂O and H₂O. The refractive index difference between D₂O and H₂O was shown to be a minor contribution by means of experiments in which D₂O was at 5 dgC and H₂O at 47 dgC, conditions of equal refractive index (Na_D). D₂O vs H₂O and glycerol-d vs glycerol-h difference spectra of ribonuclease are about twice as large as expected from the known number of exposed tyrosyl side chains. Possible sources of error in D₂O vs H₂O spectra of proteins are discussed.