Bernd Voigt

Fluorescence of native and carotenoid-depleted LH2 from Chromatium minutissimum, originating from simultaneous two-photon absorption in the spectral range of the presumed (optically ‘dark’) S1 state of carotenoids

Native and carotenoid‐depleted peripheral purple bacterial light‐harvesting complex (LH2) were investigated by simultaneous two‐photon excited (between 1300–1500 nm) fluorescence (TPF). TPF results from direct bacteriochlorophyll excitation in both samples. The spectral position of the 2Ag − state of rhodopsin is indicated by a diminuition of the bacteriochlorophyll TPF in native LH2. In conclusion, comparison to carotenoid‐depleted samples is a conditio sine qua non for unambiguous interpretation of similar experiments.

Excitonic Coupling of Chlorophylls in the Plant Light-Harvesting Complex LHC-II

Manifestation and extent of excitonic interactions in the red Chl-absorption region (Q(y) band) of trimeric LHC-II were investigated using two complementary nonlinear laser-spectroscopic techniques. Nonlinear absorption of 120-fs pulses indicates an increased absorption cross section in the red wing of the Q(y) band as compared to monomeric Chl a in organic solution. Additionally, the dependence of a nonlinear polarization response on the pump-field intensity was investigated. This approach reveals that one emitting spectral form, characterized by a 2.3(+/-0.8)-fold larger dipole strength than monomeric Chl a, dominates the fluorescence spectrum of LHC-II. Considering available structural and spectroscopic data, these results can be consistently explained assuming the existence of an excitonically coupled dimer located at Chl-bindings sites a2 and b2 (referring to the original notation of W. Nühlbrandt, D.N. Wang, and Y. Fujiyoshi, Nature, 1994, 367:614-621), which must not necessarily correspond to Chls a and b). This fluorescent dimer, terminating the excitation energy-transfer chain of the LHC-II monomeric subunit, is discussed with respect to its relevance for intra- and inter-antenna excitation energy transfer.

NONLINEAR POLARIZATION SPECTROSCOPY (FREQUENCY DOMAIN) STUDIES OF EXCITED STATE PROCESSES: THE B800–850ANTENNA OF RHODOBACTER SPHAEROIDES

Nonlinear polarization spectroscopy in the frequency domain allows rate constant determinations of fast electronic energy and phase relaxations together with characterization of the type of line broadening. Application of this method to the B850 component of the isolated B800–850antenna ofRhodobacter sphaeroides at room temperature shows that B850 is inhomogeneously broadened, with homogeneous widths between 30 and 200 cm−1, depending on the spectral position of the subforms. The corresponding phase relaxation times are clearly in the subpicosecond range. There is also indication of an up‐to‐now unspecified1–5 ps energy relaxation channel per subunit.

Pigment–protein architecture in the light-harvesting antenna complexes of purple bacteria: does the crystal structure reflect the native pigment–protein arrangement?

Structural analysis of crystallized peripheral (LH2) and core antenna complexes (LH1) of purple bacteria has revealed circular aggregates of high rotational symmetry (C8, C9 and C16, respectively). Quantum‐chemical calculations indicate that in particular the waterwheel‐like arrangements of pigments should show characteristic structure‐sensitive spectroscopic behavior in the near infrared absorption region. Laser‐spectroscopic data obtained with non‐crystallized, isolated LH2 of Rhodospirillum molischianum are in line with a highly symmetric (C8) circular aggregate, but deviations have been found for LH2 of Rhodobacter sphaeroides and Rhodopseudomonas acidophila. For both the latter, C‐shaped incomplete circular aggregates (as seen only recently in electron micrographs of crystallized LH1–reaction center complexes) may be a suitable preliminary model.

Direct evidence for excitonically coupled chlorophylls a and b in LHC II of higher plants by nonlinear polarization spectroscopy in the frequency domain

Occurrence of excitonic interactions in light-harvesting complex II (LHC II) was investigated by nonlinear polarization spectroscopy in the frequency domain (NLPF) at room temperature. NLPF spectra were obtained upon probing in the chlorophyll (Chl) a/b Soret region and pumping in the Q(y) region. The lowest energy Chl a absorbing at 678 nm is strongly excitonically coupled to Chl b.

A new set-up for nonlinear polarization spectroscopy in the frequency domain: experimental examples and theoretical background

The advantages of a new experimental geometry for nonlinear polarization spectroscopy in the frequency domain (NLPF) are demonstrated. First, homogeneous illumination of the sample allows one to use lower pump intensities, so that the approach to describing the NLPF signal is valid. Secondly, there being fewer artefacts from competitive scattering processes results in distortionless NLPF spectra. These experimental improvements are illustrated by experimental results concerning pinacyanol.

Low-intensity two-step absorption of chlorophyll A in vivo

The intensity-dependent transmission of primary leaves of Triticum aestivum seedlings at lambda = 694 nm was measured with single pulses of a Q-switch ruby laser. At photon flux densities above 2 x 10(17) cm-2s-1 a decrease of transmission was observed. The result is interpreted as a two-step absorption of cooperative units of 10(5)-10(6) chlorophyll molecules.

Direct Resolution of Spectral Fine-Structure and Ultrafast Exciton Dynamics in Light Harvesting Complex II by Nonlinear Polarization Spectroscopy in the Frequency Domain

In plants efficient light capture and highly regulated excitation energy transfer to the reaction centers is assured by chlorophyll (Chl) a/b containing light-harvesting complexes (LHCs) [1]. The structure of the major (mainly photosystem II-associated) complex (LHC II) is known to 3.4 A resolution [2] — but the reflection of the structure in optical spectra/exciton dynamics is not fully understood, yet. Upon aggregation LHC undergoes considerable changes of its spectroscopic properties — most prominent a pronounced quenching of Chl-fluorescence. Thus it has been suggested that reversible aggregation of LHC II may establish the molecular basis of “high-energy quenching” (qE) — an important photo-protective mechanism in plants [3–5]. Both phenomena, LHC aggregation and qE have been found to be associated with red-shifted Chl spectral species [3,6]. We employ Nonlinear Polarization Spectroscopy in the Frequency Domain (NLPF) — a four-wave mixing technique (Fig. 1) — to investigate spectral sub-structure, band-broadening and ps/fs exciton dynamics in trimeric and aggregated LHC II [7,8].

Non-Linear Spectroscopy of Carotenoid-Chlorophyll Interactions in Photosynthetic Light-Harvesting Complexes

In addition to chlorophylls a and b light-harvesting complex (LHC II) binds xanthophylls. Nonlinear polarization spectroscopy in the frequency domain (NLPF) was used to investigate the changes in the interactions between xanthophylls and chlorophylls in LHC II upon alteration of its aggregation state. Additionally, two-photon excitation profiles in the xanthophylls presumed 11Ag − → 21Ag − transition region were measured of LHC II samples containing different xanthophyll complements, as well as of chlorophylls a and b in solution. Implications of the results for recently proposed mechanism(s) of qE/NPQ will be discussed.

Two-photon fluorescence excitation spectroscopy of photosynthetic pigments and pigment-protein complexes

Chlorophylls (Chls) and carotenoids are light-harvesting pigments and essential structural components of photosynthetic pigment-protein complexes. Due to the optically forbidden character of the lowest excited singlet state (S<inf>1</inf>/2¹A<inf>g</inf>⁻) for one-photon excitation from the electronic ground state (S<inf>0</inf>/1¹A<inf>g</inf>⁻) of relevant carotenoids, the relative energetic position of the carotenoids S<inf>1</inf> state cannot be readily investigated by conventional spectroscopic techniques. In several light-harvesting complexes (LHCs) this state, however, is assumed to be involved in excitation energy transfer to adjacent Chl or bacteriochlorophyll (BChl) molecules. This is based on a supposed close energetic proximity of the carotenoid S<inf>1</inf> state to the Chl or BChl S<inf>1</inf> (Q<inf>y</inf>) state. The carotenoid S<inf>0</inf> to S<inf>1</inf>-transition is two-photon allowed. Consequently, peaks in the two-photon fluorescence (TPF) excitation spectra of LHCs (detected by Chl- or BChl-fluorescence) have usually been ascribed to this transition [1].