Marta Germano

Supramolecular organization of photosystem I and light- harvesting complex I in Chlamydomonas reinhardtii

We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of photosystem I and light‐harvesting complex I from the unicellular green alga Chlamydomonas reinhardtii. The complex is a monomer, has longest dimensions of 21.3 and 18.2 nm in projection, and is significantly larger than the corresponding complex in spinach. Comparison with photosystem I complexes from other organisms suggests that the complex contains about 14 light‐harvesting proteins, two or three of which bind at the side of the PSI‐H subunit. We suggest that special light‐harvesting I proteins play a role in the binding of phosphorylated light‐harvesting complex II in state 2.

Photosystem II solubilizes as a monomer by mild detergent treatment of unstacked thylakoid membranes

We studied the aggregation state of Photosystem II in stacked and unstacked thylakoid membranes from spinach after a quick and mild solubilization with the non-ionic detergent n-dodecyl-α,D-maltoside, followed by analysis by diode-array-assisted gel filtration chromatography and electron microscopy. The results suggest that Photosystem II (PS II) isolates either as a paired, appressed membrane fragment or as a dimeric PS II-LHC II supercomplex upon mild solubilization of stacked thylakoid membranes or PS II grana membranes, but predominantly as a core monomer upon mild solubilization of unstacked thylakoid membranes. Analysis of paired grana membrane fragments reveals that the number of PS II dimers is strongly reduced in single membranes at the margins of the grana membrane fragments. We suggest that unstacking of thylakoid membranes results in a spontaneous disintegration of the PS II-LHC II supercomplexes into separated PS II core monomers and peripheral light-harvesting complexes.

Energy and Electron Transfer in Photosystem II Reaction Centers with Modified Pheophytin Composition

Energy and electron transfer in Photosystem II reaction centers in which the photochemically inactive pheophytin had been replaced by 13(1)-deoxo-13(1)-hydroxy pheophytin were studied by femtosecond transient absorption-difference spectroscopy at 77 K and compared to the dynamics in untreated reaction center preparations. Spectral changes induced by 683-nm excitation were recorded both in the Q(Y) and in the Q(X) absorption regions. The data could be described by a biphasic charge separation. In untreated reaction centers the major component had a time constant of 3.1 ps and the minor component 33 ps. After exchange, time constants of 0.8 and 22 ps were observed. The acceleration of the fast phase is attributed in part to the redistribution of electronic transitions of the six central chlorin pigments induced by replacement of the inactive pheophytin. In the modified reaction centers, excitation of the lowest energy Q(Y) transition produces an excited state that appears to be localized mainly on the accessory chlorophyll in the active branch (B(A) in bacterial terms) and partially on the active pheophytin H(A). This state equilibrates in 0.8 ps with the radical pair. B(A) is proposed to act as the primary electron donor also in untreated reaction centers. The 22-ps (pheophytin-exchanged) or 33-ps (untreated) component may be due to equilibration with the secondary radical pair. Its acceleration by H(B) exchange is attributed to a faster reverse electron transfer from B(A) to. After exchange both and are nearly isoenergetic with the excited state.

Selective replacement of the active and inactive pheophytin in reaction centres of Photosystem II by 13(1)-deoxo-13(1)-hydroxy-pheophytin a and comparison of their 6 K absorption spectra.

Pheophytin a (Pheo) in Photosystem II reaction centres was exchanged for 131-deoxo-131-hydroxy-pheophytin a (131-OH-Pheo). The absorption bands of 131-OH-Pheo are blue-shifted and well separated from those of Pheo. Two kinds of modified reaction centre preparations can be obtained by applying the exchange procedure once (RC1×) or twice (RC2×). HPLC analysis and Pheo QX absorption at 543 nm show that in RC1× about 50% of Pheo is replaced and in RC2× about 75%. Otherwise, the pigment and protein composition are not modified. Fluorescence emission and excitation spectra show quantitative excitation transfer from the new pigment to the emitting chlorophylls. Photoaccumulation of Pheo− is unmodified in RC1× and decreased only in RC2×, suggesting that the first exchange replaces the inactive and the second the active Pheo. Comparing the effects of the first and the second replacement on the absorption spectrum at 6 K did not reveal substantial spectral differences between the active and inactive Pheo. In both cases, the absorption changes in the QY region can be interpreted as a combination of a blue shift of a transition at 684 nm, a partial decoupling of chlorophylls absorbing at 680 nm and a disappearance of Pheo absorption in the 676-680 nm region. No absorption decrease is observed at 670 nm for RC1× or RC2×, showing that neither of the two reaction centre pheophytins contributes substantially to the absorption at this wavelength.

ENERGY TRANSFER AND TRAPPING IN PHOTOSYSTEM II CORE PARTICLES WITH CLOSED REACTION CENTERS

Picosecond absorbance changes in the Qy absorption region were measured on Photosystem II core particles with closed reaction centers by the one-color pump-probe method. The induced absorption changes are well described by three components with lifetimes of 21 ± 6 ps, 80 to 200 ps and 1.5 ns, in addition to a non-decaying component. The 1.5 ns lifetime component is assigned to recombination of the primary radical pair in equilibrium with the excited state. Since the lifetime of the intermediate component depends on the excitation wavelength and its spectrum differs from that of the 21 ps component, a rapid equilibration of the excitations over the whole antenna is excluded. The 21 ps and the intermediate component are discussed in terms of energy transfer and trapping processes.

Triplet state in photosystem II reaction centers as studied by 130 GHz EPR

Abstract The triplet state in the reaction centers of photosystem II was studied by high-field/high-frequency (130 GHz) EPR in the temperature range 50–90 K. At 50 K, the zero-field splitting parameters of the EPR spectrum correspond well to those of a chlorophyll monomer, in agreement with earlier studies. In the high magnetic field of 4.6 T employed in this study, the g-anisotropy of the triplet state becomes apparent and leads to a shift of the canonical positions of the triplet EPR spectrum. Assuming that triplet g- and zero-field tensors are coaxial, the principal values of the triplet g-tensor are determined to be 2.00324, 2.00306 and 2.00231 with an error of ±0.00004. Lifting this assumption results in higher g-anisotropy. At higher temperatures, the shape of the spectra changes significantly. Triplet excitation hopping involving the accessory chlorophyll BA and PA or PB (equivalents of the special pair bacteriochlorophylls of the bacterial reaction centers) can partially explain those changes, but the most prominent features indicate that also the electron acceptor IA (a pheophytin molecule) must be involved.