F. van Mourik

Direct observation of sub-picosecond equilibration of excitation energy in the light-harvesting antenna of Rhodospirillum rubrum.

Excitation energy transfer in the light-harvesting antenna of Rhodospirillum rubrum was studied at room temperature using sub-picosecond transient absorption measurements. Upon excitation of Rs. rubrum membranes with a 200 fs, 600 nm laser flash in the Qx transition of the bacteriochlorophyll-a (BChl-a) absorption, the induced transient absorption changes in the Qy region were monitored. In Rs. rubrum membranes the observed delta OD spectrum exhibits ground state bleaching, excited state absorption and stimulated emission. Fast Qx --> Qy relaxation occurs in approximately 100-200 fs as reflected by the building up of stimulated emission. An important observation is that the zero-crossing of the transient difference absorption (delta OD) spectrum exhibits a dynamic redshift from 863 to 875 nm that can be described with by a single exponential with 325 fs time constant. The shape of the transient difference spectrum observed in a purified subunit of the core light-harvesting antenna, B820, consisting of only a single interacting pair of BChl-as, is similar to the spectrum observed in Rs. rubrum membranes and clearly different from the spectrum of BChl-a in a protein/detergent mixture. In the B820 and monomeric BChl-a preparations the 100-200 fs Qx --> Qy relaxation is still observed, but the dynamic redshift of the delta OD spectrum is absent. The spectral kinetics observed in the Rs. rubrum membranes are interpreted in terms of the dynamics of excitation equilibration among the antenna subunits that constitute the inhomogeneously broadened antenna. A simulation of this process using a set of reasonable physical parameters is consistent with an average hopping time in the core light harvesting of 220-270 fs, resulting in an average single-site excitation lifetime of 50-70 fs. The observed rate of this equilibration process is in reasonable agreement with earlier estimations for the hopping time from more indirect measurements. The implications of the findings for the process of excitation trapping by reaction centers will be discussed.

Energy transfer and aggregate size effects in the inhomogeneously broadened core light-harvesting complex of Rhodobacter sphaeroides

Abstract The 4 K fluorescence emission spectrum of the core light-harvesting complex of the photosynthetic purple bacterium Rhodobacter sphaeroides is shown to depend strongly on the wavelength of excitation. It is concluded that the dominant factor determining the absorption spectrum is inhomogeneous broadening. The site-selected emission spectra are interpreted in terms of energy transfer in a cluster of weakly coupled pigments. Each individual cluster is supposed to be a random sample from the total inhomogeneously broadened pigment pool. For small clusters, with efficient energy transfer leading to fast thermalization, the effects of site-selection depend on the cluster-size only. It is shown that site-selective excitation can yield information about the number of coupled pigments in such a cluster. For the purified LH-1 of Rb. sphaeroides the cluster size was estimated to be 16 pigments.

(Sub)-Picosecond Spectral Evolution of Fluorescence in Photoactive Proteins Studied with a Synchroscan Streak Camera System

Abstract The spectral evolution of three photoactive proteins has been investigated by measuring the fluorescence with good temporal and wavelength resolution and a high signal-to-noise ratio. Upon excitation at 400 nm wild-type (wt) PYP both at neutral pH and in the low-pH blueshifted pBdark state exhibited a strong quenching of the fluorescence, the major part of which could be described by lifetimes of about 1.7 and 7.7 ps. The remaining fluorescence decay occurred multiexponentially with lifetimes between 30 and 125 ps. Additionally, in wtPYP at neutral pH, a dynamic Stokes shift was found to occur with a time constant of about 0.25 ps. In a PYP preparation that was reconstituted with the chromophore 7-hydroxy-coumarin-3- carboxylic acid rather than the native coumaric acid, and which is therefore not capable of performing the cis-trans-isomerization that initiates the photocycle in wtPYP, the fluorescence was found to decay multiexponentially with lifetimes of 51 ps, 0.33 and 3.77 ns. Additionally, dynamic Stokes shifts were observed with time constants of about 0.1 and 3.5 ps. Upon comparison of the dynamics of this preparation with that of wtPYP the multiexponential decay with lifetimes of 1.7 and 7.7 ps found in wtPYP was attributed to photochemistry of the p-coumaric-acid chromophore. The emission from bacteriorhodopsin mutant D85S upon excitation at 635 nm decays biexponentially with estimated lifetimes of 5.2 and 19.1 ps. No dynamic Stokes shift was observed here. Four lifetimes were needed to describe the decay of the emission from the A* state in the green fluorescent protein. From a target analysis it was concluded that the longer lifetimes are accompanied by a decreasing probability of forming I*, which approaches zero with the longest A* lifetime of 1.5 ns. These observations may be explained by heterogeneity of A and by relaxation of A*. In all three systems studied, multiexponential decay of emission was present, suggesting that heterogeneity is a common feature of these chromophore protein complexes.

Electron-phonon coupling in the B820 subunit form of LH1 studied by temperature dependence of optical spectra

Abstract The temperature dependence of absorption and selectively excited fluorescence spectra of B820 subunit of the bacterial light-harvesting antenna is modelled by using the impurity center theory ofoptical spectra. In order toget acceptable agreement between theory and experiment, we suggest that due to the dimeric nature of B820, the electronic transition is weakly coupled to a rather broad distribution of protein phonons.

Functional electric field changes in photoactivated proteins revealed by ultrafast Stark spectroscopy of the Trp residues

Ultrafast transient absorption spectroscopy of wild-type bacteriorhodopsin (WT bR) and 2 tryptophan mutants (W86F and W182F) is performed with visible light excitation (pump) and UV probe. The aim is to investigate the photoinduced change in the charge distribution with 50-fs time resolution by probing the effects on the tryptophan absorption bands. A systematic, quantitative comparison of the transient absorption of the 3 samples is carried out. The main result is the absence in the W86F mutant of a transient induced absorption band observed at ≈300–310 nm in WT bR and W182F. A simple model describing the dipolar interaction of the retinal moiety with the 2 tryptophan residues of interest allows us to reproduce the dominant features of the transient signals observed in the 3 samples at ultrashort pump-probe delays. In particular, we show that Trp86 undergoes a significant Stark shift induced by the transient retinal dipole moment. The corresponding transient signal can be isolated by direct subtraction of experimental data obtained for WT bR and W86F. It shows an instantaneous rise, followed by a decay over ≈500 fs corresponding to the isomerization time. Interestingly, it does not decay back to zero, thus revealing a change in the local electrostatic environment that remains long after isomerization, in the K intermediate state of the protein cycle. The comparison of WT bR and W86F also leads to a revised interpretation of the overall transient UV absorption of bR.

Spectroscopy and structure of bacteriochlorophyll dimers. I. Structural consequences of nonconservative circular dichroism spectra.

The origin of the nonconservative nature of the circular dichroism (CD) spectrum of bacteriochlorophyll dimers is investigated. It is shown that coupling between the Qy and Qx transitions can, under rather restricting circumstances, lead to an asymmetrical CD spectrum: only for a limited set of relative orientations of the monomers within the dimer is the spectrum found to be asymmetrical. The relation between intensity and asymmetry of the CD spectrum is elucidated. The results are applied to the B820 subunit of the LH1 antenna system and subsequently to the antenna system LH1 itself. Differences in the geometry of the BChls in LH1 versus the LH2 structure are discussed.

Relaxation dynamics of tryptophan in water: A UV fluorescence up-conversion and molecular dynamics study.

We report on an ultrafast experimental and simulations study of the early relaxation events of photoexcited tryptophan in water. Experimentally, we used fluorescence up-conversion in both polychromatic and single wavelength detection modes in the 300-480 nm range with polarization dependence. We report on the time evolution of the Stokes shift, bandwidth, and anisotropy from tens of femtoseconds to picoseconds. These observables contain signatures of the simultaneous occurrence of intramolecular and solvent-molecule interactions, which we disentangle with the help of nonequilibrium molecular dynamics simulations. We also observe a breakdown of the linear response approximation to describe our results.

Harmonium: A pulse preserving source of monochromatic extreme ultraviolet (30–110 eV) radiation for ultrafast photoelectron spectroscopy of liquids

A tuneable repetition rate extreme ultraviolet source (Harmonium) for time resolved photoelectron spectroscopy of liquids is presented. High harmonic generation produces 30–110 eV photons, with fluxes ranging from ∼2 × 1011 photons/s at 36 eV to ∼2 × 108 photons/s at 100 eV. Four different gratings in a time-preserving grating monochromator provide either high energy resolution (0.2 eV) or high temporal resolution (40 fs) between 30 and 110 eV. Laser assisted photoemission was used to measure the temporal response of the system. Vibrational progressions in gas phase water were measured demonstrating the ∼0.2 eV energy resolution.

Linear dichroism of the complex between the gene 32 protein of bacteriophage T4 and poly(1,N6-ethenoadenylic acid)

We performed linear dichroism measurements in compressed polyacrylamide gels on the complex between the helix-destabilizing protein of bacteriophage T4, GP32 and poly(1,N6-ethenoadenylic acid), which is used as a model system for single-stranded DNA. A strong hyperchromism for poly(1,N6-ethenoadenylic acid) in the complex indicates a strongly altered conformation. The positive linear dichroism in the wavelength region where the bases absorb must be explained by a strong tilting of the bases in the complex. This finding is in accordance with results from earlier studies, using electric birefringence and circular dichroism measurements. Our measurements show that the angle between the bases and the local helix axis is 42(+/- 6)degrees. In addition, a pronounced contribution from the tryptophan residues of GP32 can be recognized, indicating that several of these residues have a specific orientation in the complex. The sign of the dichroism due to the tryptophan residues is the same as that due to the DNA bases. However, it is not sufficient to assume that all the observed dichroism is due to one or more intercalated tryptophan residues and there must be one or more additional tryptophan residues that make an angle of less than 40 degrees with the local helix axis. Some possible structures of the DNA-protein complex are discussed.

The internal dynamics of gene 32 protein-DNA complexes studied by quasi-elastic light scattering

The hydrodynamic properties of large homodisperse single stranded DNAs complexed with the helix destabilizing protein of phage T4, the product of gene 32 (GP32), have been measured. The results suggest a size of the binding site between 8 and 10 nucleotides/GP32 molecule, in reasonable agreement with earlier work on a complex between GP32 and single stranded 145 base DNA. From static light scattering experiments it is concluded that the persistence length of these complexes is about 30 nm, distinctly smaller than the generally accepted value for double stranded DNA. The quasi-elastic light scattering properties of the DNA-GP32 complexes were determined. The variation of the apparent translation diffusion coefficient Dapp with the scattering vector q was analyzed using the discrete ISMF and Rouse-Zimm models [S.C. Lin et al., Biopolymers 17 (1978) 425]. The model parameters that followed from the fit of Dapp versus q2 and from an extensive global analysis of the actually measured autocorrelation functions agreed with the notion that these DNA-protein complexes are indeed rather flexible. The continuous Soda model [K. Soda, Macromolecules 17 (1984) 2365] could successfully explain the variation of Dapp versus q2, assuming a persistence length of 30 nm and a base-base distance in the complex of 0.44 nm.