Klaus-Dieter Irrgang

Crystal structure of cyanobacterial photosystem II at 3.2 Å resolution: a closer look at the Mn-cluster

In the crystal structure of photosystem II (PSII) from the cyanobacterium Thermosynechococcus elongatus at 3.2 A resolution, several loop regions of the principal protein subunits are now defined that were not interpretable previously at 3.8 A resolution. The head groups and side chains of the organic cofactors of the electron transfer chain and of antenna chlorophyll a (Chl a) have been modeled, coordinating and hydrogen bonding amino acids identified and the nature of the binding pockets derived. The orientations of these cofactors resemble those of the reaction center from anoxygenic purple bacteria, but differences in hydrogen bonding and protein environment modulate their properties and provide the unique high redox potential (1.17 V) of the primary donor. Coordinating amino acids of manganese cluster, redox-active TyrZ and non-haem Fe2+ have been determined, and an all-trans β-carotene connects cytochrome b-559, ChlZ and primary electron donor (coordinates are available under PDB-code 1W5C).

Assignment of the Lowest QY-state and Spectral Dynamics of the CP29 Chlorophyll a/b Antenna Complex of Green Plants: A Hole-burning Study ‡

Abstract Low-temperature absorption, fluorescence and persistent nonphotochemical hole-burned spectra are reported for the CP29 chlorophyll (Chl) a/b antenna complex of photosystem II of green plants. The absorption-origin band of the lowest Qy-state lies at 678.2 nm and carries a width of ∼130 cm−1 that is dominated by inhomogeneous broadening at low temperatures. Its absorption intensity is equivalent to that of one of the six Chl a molecules of CP29. The absence of a significant satellite hole structure produced by hole burning, within the absorption band of the lowest state, indicates that the associated Chl a molecule is weakly coupled to the other Chl and, therefore, that the lowest-energy state is highly localized on a single Chl a molecule. The electron–phonon coupling of the 678.2 nm state is weak with a Huang–Rhys factor S of 0.5 and a peak phonon frequency (ωm) of ∼20 cm−1. These values give a Stokes shift (2Sωm) in good agreement with the measured positions of the absorption band at 678.2 nm and a fluorescence-origin band at 679.1 nm. Zero-phonon holes associated with the lowest state have a width of ∼0.05 cm−1 at 4.2 K, corresponding to a total effective dephasing time of ∼400 ps. The temperature dependence of the zero-phonon holewidth indicates that this time constant is dominated at temperatures below 8 K by pure dephasing/spectral diffusion due to coupling of the optical transition to the glass-like two-level systems of the protein. Zero-phonon holewidths obtained for the Chl b bands at 638.5 and 650.0 nm, at 4.2 K, lead to lower limits of 900 ± 150 fs and 4.2 ± 0.3 ps, respectively, for the Chl b → Chl a energy-transfer times. Downward energy transfer from the Chl a state(s) at 665.0 nm occurs in 5.3 ± 0.6 ps at 4.2 K.

Chromophore-chromophore and chromophore-protein interactions in monomeric light-harvesting complex II of green plants studied by spectral hole burning and fluorescence line narrowing.

Persistent nonphotochemical hole burning and delta-FLN spectra obtained at 4.5 K are reported for monomeric chlorophyll (Chl) a/b light-harvesting complexes of photosystem II (LHC II) of green plants. The hole burned spectra of monomeric LHC II appear to be similar to those obtained before for trimeric LHC II (Pieper et al. J. Phys. Chem. B 1999, 103, 2412). They are composed of three main features: (i) a homogeneously broadened zero-phonon hole coincident with the burn wavelength, (ii) an intense, broad hole in the vicinity of approximately 680 nm as a result of efficient excitation energy transfer to a low-energy trap state, and (iii) a satellite hole at approximately 649 nm which is correlated with the low-energy 680 nm hole. Zero-phonon hole action spectroscopy reveals that the low-energy absorption band is located at 679.6 nm and possesses a width of approximately 110 cm(-1) which is predominantly due to inhomogeneous broadening at 4.5 K. The electron-phonon coupling of the above-mentioned low-energy state(s) is weak with a Huang-Rhys factor S in the order of 0.6 and a peak phonon frequency (omega(m)) of approximately 22 cm(-1) within a broad and strongly asymmetric one-phonon profile. The resulting Stokes shift 2S omega(m) of approximately 26.4 cm(-1) readily explains the position of the fluorescence origin band at 680.8 nm. Thus, we conclude that the 679.6 nm state(s) is (are) the fluorescent state(s) of monomeric LHC II at 4.5 K. The absorption intensity of the lowest Q(y) state is shown to roughly correspond to that of one out of the eight Chl a molecules bound in the monomeric subunit. In addition, the satellite hole structure produced by hole burning within the 679.6 nm state is weak with only one shallow satellite hole observed in the Chl b spectral range at 648.8 nm. These results suggest that the 679.6 nm state is widely localized on a Chl a molecule, which may belong to a Chl a/b heterodimer. These characteristics are different from those expected for Chl a612, which has been associated with the fluorescent state at room temperature. Alternatively, the 679.6 nm state may be assigned to Chl a604, which is located in a cluster with several Chl b molecules resulting in a relatively weak excitonic coupling.

Parameters of the protein energy landscapes of several light-harvesting complexes probed via spectral hole growth kinetics measurements.

The parameters of barrier distributions on the protein energy landscape in the excited electronic state of the pigment/protein system have been determined by means of spectral hole burning for the lowest-energy pigments of CP43 core antenna complex and CP29 minor antenna complex of spinach Photosystem II (PS II) as well as of trimeric and monomeric LHCII complexes transiently associated with the pea Photosystem I (PS I) pool. All of these complexes exhibit sixty to several hundred times lower spectral hole burning yields as compared with molecular glassy solids previously probed by means of the hole growth kinetics measurements. Therefore, the entities (groups of atoms), which participate in conformational changes in protein, appear to be significantly larger and heavier than those in molecular glasses. No evidence of a small (∼1 cm(-1)) spectral shift tier of the spectral diffusion dynamics has been observed. Therefore, our data most likely reflect the true barrier distributions of the intact protein and not those related to the interface or surrounding host. Possible applications of the barrier distributions as well as the assignments of low-energy states of CP29 and LHCII are discussed in light of the above results.

Identification of Chl-binding proteins in a PS II preparation from spinach

Using a mild solubilization procedure we have been able to detect several Chl‐binding proteins by SDS‐urea‐PAGE. Their molecular masses were 46, 42, 34, 32, 29‐24 and approx. 110 kDa (presumably a CPI contamination). The stability of the Chl‐protein complexes depended strongly on experimental conditions. A very faint band at 34‐30 kDa was resolved in visible light (in 40–50% of the experiments). With a more sensitive fluorometric technique (illumination at 366 nm, fluorescence detection in the red) we identified two Chlcontaining bands in the 30 kDa region. Limited proteolysis studies with trypsin revealed that the extent of the enzymatic degradation of some Chl‐binding proteins in vitro is obviously dependent on pH and the presence of Ca2+.

Identification, isolation and partial characterisation of a 14–15 kDa pigment binding protein complex of PS II from spinach

Abstract We described recently a new Chl a b binding polypeptide in PS II from spinach (Irrgang, K.-D., Bechtel, C., Vater, J. and Renger, G. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., ed.), Vol. I, pp. 375–378, Kluwer, Dordrecht). This 14–15 kDa chlorophyll a b binding polypeptide complex was isolated and purified from intrinsic photosystem II membrane polypeptides. About 3 Chl a and 1 Chl b were bound per protein molecule. A polyclonal antiserum was induced against this pigment-protein complex. SDS polyacrylamide gradient gel electrophoresis in combination with immunoblotting revealed reactivities with two polypeptides of very similar relative molecular masses of 14 and 15 kDa that are clearly identified in thylakoids, PS II membrane fragments, ISO and RSO thylakoids. Their N-termini were blocked by an as yet unidentified modifying group. Using analytical isoelectric focusing under denaturing conditions their isoelectric points were determined to be 5.2–5.3 and around 6.0–6.5. The isolated polypeptides of the pigment-protein complex tend to self-associate into oligomeric forms of about 66–70 kDa. Furthermore, under mild solubilisation conditions an oligomeric pigment-protein complex of 120 kDa was observed. This oligomer was shown to be heterogeneously composed of the 14–15 kDa proteins and at least another pigment-binding polypeptide with an Mr of 22–24 kDa. The low molecular mass pigment-protein complex (CP14–15) is proposed to act as an additional antenna complex within PS II.

Protein Dynamics Tunes Excited State Positions in Light-Harvesting Complex II

Light harvesting and excitation energy transfer in photosynthesis are relatively well understood at cryogenic temperatures up to ∼100 K, where crystal structures of several photosynthetic complexes including the major antenna complex of green plants (LHC II) are available at nearly atomic resolution. The situation is much more complex at higher or even physiological temperatures, because the spectroscopic properties of antenna complexes typically undergo drastic changes above ∼100 K. We have addressed this problem using a combination of quasielastic neutron scattering (QENS) and optical spectroscopy on native LHC II and mutant samples lacking the Chl 2/Chl a 612 pigment molecule. Absorption difference spectra of the Chl 2/Chl a 612 mutant of LHC II reveal pronounced changes of spectral position and their widths above temperatures as low as ∼80 K. The complementary QENS data indicate an onset of conformational protein motions at about the same temperature. This finding suggests that excited state positions in LHC II are affected by protein dynamics on the picosecond time scale. In more detail, this means that at cryogenic temperatures the antenna complex is trapped in certain protein conformations. At higher temperature, however, a variety of conformational substates with different spectral position may be thermally accessible. At the same time, an analysis of the widths of the absorption difference spectra of Chl 2/Chl a 612 reveals three different reorganization energies or Huang-Rhys factors in different temperature ranges, respectively. These findings imply that (dynamic) pigment-protein interactions fine-tune electronic energy levels and electron-phonon coupling of LHC II for efficient excitation energy transfer at physiological temperatures.

Stepwise Two-photon Excited Fluorescence from Higher Excited States of Chlorophylls in Photosynthetic Antenna Complexes

Stepwise two-photon excited fluorescence (TPEF) spectra of the photosynthetic antenna complexes PCP, CP47, CP29, and light-harvesting complex II (LHC II) were measured. TPEF emitted from higher excited states of chlorophyll (Chl) a and b was elicited via consecutive absorption of two photons in the Chl a/b Qy range induced by tunable 100-fs laser pulses. Global analyses of the TPEF line shapes with a model function for monomeric Chl a in a proteinaceous environment allow distinction between contributions from monomeric Chls a and b, strongly excitonically coupled Chls a, and Chl a/b heterodimers/-oligomers. The analyses indicate that the longest wavelength-absorbing Chl species in the Qy region of LHC II is a Chl a homodimer with additional contributions from adjacent Chl b. Likewise, in CP47 a spectral form at ∼680 nm (that is, however, not the red-most species) is also due to strongly coupled Chls a. In contrast to LHC II, the red-most Chl subband of CP29 is due to a monomeric Chl a. The two Chls b in CP29 exhibit marked differences: a Chl b absorbing at ∼650 nm is not excitonically coupled to other Chls. Based on this finding, the refractive index of its microenvironment can be determined to be 1.48. The second Chl b in CP29 (absorbing at ∼640 nm) is strongly coupled to Chl a. Implications of the findings with respect to excitation energy transfer pathways and rates are discussed. Moreover, the results will be related to most recent structural analyses.

A New Chl a/b Binding Protein in Photosystem II from Spinach with a Mr of 14 kDa

It has been widely accepted that in higher plants all the pigments that are involved in the photosynthetic primary processes are bound to thylakoid membrane polypeptides. Among these pigment-binding proteins about the half belong to the so-called Chl a/b light-harvesting antennae. With respect to photosystem II we know at least four complexes with relative molecular masses of 29 (CP 29), 27 (CP27), 25 (major LHC II) and 24 (CP24) kDa (1–4).