Solution structure and excitation energy transfer in phycobiliproteins of Acaryochloris marina investigated by small angle scattering.

The structure of phycobiliproteins of the cyanobacterium Acaryochloris marina was investigated in buffer solution at physiological temperatures, i.e. under the same conditions applied in spectroscopic experiments, using small angle neutron scattering. The scattering data of intact phycobiliproteins in buffer solution containing phosphate can be well described using a cylindrical shape with a length of about 225Å and a diameter of approximately 100Å. This finding is qualitatively consistent with earlier electron microscopy studies reporting a rod-like shape of the phycobiliproteins with a length of about 250 (M. Chen et al., FEBS Letters 583, 2009, 2535) or 300Å (J. Marquart et al., FEBS Letters 410, 1997, 428). In contrast, phycobiliproteins dissolved in buffer lacking phosphate revealed a splitting of the rods into cylindrical subunits with a height of 28Å only, but also a pronounced sample aggregation. Complementary small angle neutron and X-ray scattering experiments on phycocyanin suggest that the cylindrical subunits may represent either trimeric phycocyanin or trimeric allophycocyanin. Our findings are in agreement with the assumption that a phycobiliprotein rod with a total height of about 225Å can accommodate seven trimeric phycocyanin subunits and one trimeric allophycocyanin subunit, each of which having a height of about 28Å. The structural information obtained by small angle neutron and X-ray scattering can be used to interpret variations in the low-energy region of the 4.5K absorption spectra of phycobiliproteins dissolved in buffer solutions containing and lacking phosphate, respectively.

Excitation energy transfer and electron-vibrational coupling in phycobiliproteins of the cyanobacterium Acaryochloris marina investigated by site-selective spectroscopy.

In adaption to its specific environmental conditions, the cyanobacterium Acaryochloris marina developed two different types of light-harvesting complexes: chlorophyll-d-containing membrane-intrinsic complexes and phycocyanobilin (PCB) - containing phycobiliprotein (PBP) complexes. The latter complexes are believed to form a rod-shaped structure comprising three homo-hexamers of phycocyanin (PC), one hetero-hexamer of phycocyanin and allophycocyanin (APC) and probably a linker protein connecting the PBPs to the reaction centre. Excitation energy transfer and electron-vibrational coupling in PBPs have been investigated by selectively excited fluorescence spectra. The data reveal a rich spectral substructure with a total of five low-energy electronic states with fluorescence bands at 635nm, 645nm, 654nm, 659nm and a terminal emitter at about 673 nm. The electronic states at ~635 and 645 nm are tentatively attributed to PC and APC, respectively, while an apparent heterogeneity among PC subunits may also play a role. The other fluorescence bands may be associated with three different isoforms of the linker protein. Furthermore, a large number of vibrational features can be identified for each electronic state with intense phonon sidebands peaking at about 31 to 37cm⁻¹, which are among the highest phonon frequencies observed for photosynthetic antenna complexes. The corresponding Huang-Rhys factors S fall in the range between 0.98 (terminal emitter), 1.15 (APC), and 1.42 (PC). Two characteristic vibronic lines at about 1580 and 1634cm⁻¹ appear to reflect CNH⁺ and CC stretching modes of the PCB chromophore, respectively. The exact phonon and vibrational frequencies vary with electronic state implying that the respective PCB chromophores are bound to different protein environments. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

Effect of monochromatic UV-B radiation on electron transfer reactions of Photosystem II.

The adverse effect of low intensity, small band UV-B irradiation (lambda = 305 +/- 5 nm, I = 300 mW m(-2)) on PS II has been studied by comparative measurements of laser flash-induced changes of the absorption at 325 nm, DeltaA(325)(t), as an indicator of redox changes in Q(A), and of the relative fluorescence quantum yield, F(t)/F(o), in PS II membrane fragments. The properties of untreated control were compared with those of samples where the oxygen evolution rate under illumination with continuous saturating light was inhibited by up to 95%. The following results were obtained: a) the detectable initial amplitude (at a time resolution of 30 mus) of the 325 nm absorption changes, DeltaA(325), remained virtually invariant whereas the relaxation kinetics exhibit significant changes, b) the 300 mus kinetics of DeltaA(325) dominating the relaxation in UV-B treated samples was largely replaced by a 1.3 ms kinetics after addition of MnCl(2), c) the extent of the flash induced rise of the relative fluorescence quantum yield was severely diminished in UV-B treated PS II membrane fragments but the relaxation kinetics remain virtually unaffected. Based on these results the water oxidizing complex (WOC) is inferred to be the primary target of UV-B impairment of PS II while the formation of the 'stable' radical pair P680(+*)Q(A) (-) (*) is almost invariant to this UV-B treatment.

Photoinhibition as a function of the ambient redox potential in Tris-washed PS II membrane fragments.

The mode of photoinhibition as a function of the ambient redox potential (E(ambient)) in suspensions of Tris-washed PS II membrane fragments has been analyzed by monitoring flash-induced absorption changes at 830 nm. It was found: (a) the detectable initial amplitude, DeltaA (830) (total) , as a measure of the capacity to form the 'stable' radical pair, P680(+c) Q (A) (-c) , drastically decreases during a 10 min photoinhibition at E(ambient) values below +350 mV; (b) conversely, the normalized extent of the 18 mus relaxation kinetics, DeltaA (830) (18 mus) as a measure of the electron transfer from Y(Z) to P680(+c) becomes highly susceptible to light stress when E(ambient) exceeds values of about +350 mV; (c) effects of the ambient redox potentials are highly pronounced during light exposure under anaerobic conditions, while much smaller differences arise under aerobic conditions; (d) the extent of damage does not correlate with the total concentration of K(3)[Fe(CN)(6)] and K(4)[Fe(CN)(6)] in the suspension during photoinhibition but rather depends on the E( m )-values; (e) qualitatively similar features are observed when the redox buffer system K(3)[Fe(CN)(6)]/Na(2)S(2)O(4) is replaced by K(2)[IrCl(6)]/Na(2)S(2)O(4); (f) the characteristic E(ambient)-dependence of photoinhibition is observed only under anaerobic conditions. The results are discussed with respect to different redox components that might be involved, including brief comments on a possible role of Cyt b559.

Pulsed EPR measurement of the distance between P680+. and Q(A)-. in photosystem II.

Out-of-phase electron spin echo envelope modulation (ESEEM) spectroscopy was used to determine the distance between the primary donor radical cation P680+. and the quinone acceptor radical anion Q(A)-. in iron-depleted photosystem II in membrane fragments from spinach that are deprived of the water oxidizing complex. Furthermore, a lower limit for the distance between the oxidized tyrosine residue Y(Z) of polypeptide D1 and Q(A)-. could be estimated by a comparison of data gathered from samples where the electron transfer from Y(Z) to P680+. is either intact or blocked by preillumination in the presence of NH2OH.

Effects of hydrogen/deuterium exchange on photosynthetic water cleavage in PS II core complexes from spinach.

H/D isotope exchange effects on P680+. reduction by Yz and electron abstraction from the water oxidizing complex (WOC) in redox state S3 by YZOX were analyzed in PS II core complexes from spinach by measurements of laser flash induced absorption changes at 820 nm and 355 nm. The results obtained reveal: (1) the rate of Yz oxidation by P680+. is almost independent of the substitution of exchangeable protons by deuterons; and (2) the reaction between YZOX and the WOC in S3 exhibits a kinetic H/D isotope exchange effect of similar magnitude as that recently observed in PS II membrane fragments [Renger, G., Bittner, T. and Messinger, J. (1994) Biochem. Soc. Trans. 22, 318-322]. Based on these results it is inferred that photosynthetic dioxygen formation comprises the cleavage of at least one hydrogen bond.

Flash-induced absorption spectroscopy studies of copper interaction with photosystem II in higher plants.

Measurements of flash-induced absorption changes at 325, 436, and 830 nm and of oxygen evolution were performed in order to analyze in detail the inhibition of photosystem II (PS II) by Cu(II) in PS II membrane fragments from spinach. (a) The kinetics of P680+ reduction become markedly slower in the presence of 100 microM CuSO4. (b) The CuSO4-induced kinetics of P680+ reduction are dominated by a 140-160-microsecond decay. (c) The extent of these 140-160-microsecond kinetics, normalized to the overall decay, remains virtually unaffected by addition of the exogenous PS II donor, NH2OH. (d) In thoroughly dark-adapted samples the CuSO4-induced 140-160-microsecond kinetics are already observed after the first flash and remain unchanged by a train of excitation flashes. (e) The extent of P680+ and QA- formation under repetitive flash excitation is not diminished by addition of 100 microM CuSO4. (f) The induction of microsecond kinetics of P680+ reduction at the expense of ns kinetics and the inhibition of the saturation rate of oxygen evolution exhibit the same dependence on CuSO4 concentration. (g) CuSO4 also transforms the 10-20-microsecond reduction of P680+ by TyrZ in Tris-washed PS II membrane fragments into 140-160-microsecond kinetics without any effect on the extent of flash-induced P680+ formation. These results unambiguously show that Cu(II) does not affect the charge separation (P680+QA-), but instead specifically modifies TyrZ and/or its micro environment so that the electron transfer to P680+ becomes blocked.

Functional size of Photosystem II determined by radiation inactivation.

The functional size of Photosystem II (PS II) was investigated by radiation inactivation. The technique provides an estimate of the functional mass required for a specific reaction and depends on irradiating samples with high energy γ-rays and assaying the remaining activity. The analysis is based on target theory that has been modified to take into account the temperature dependence of radiation inactivation of proteins. Using PS II enriched membranes isolated from spinach we determined the functional size of primary charge separation coupled to water oxidation and quinone reduction at the QB site: H2O → (Mn)4 → Yz → P680 → Pheophytin → Q → phenyl-p-benzoquinone. Radiation inactivation analysis indicates a functional mass of 88 ± 12 kDa for electron transfer from water to phenyl-p-benzoquinone. It is likely that the reaction center heterodimer polypeptides, D1 and D2, contribute approximately 70 kDa to the functional mass, in which case polypeptides adding up to approximately 20 kDa remain to be identified. Likely candidates are the α and ß subunits of cytochrome b 559and the 4.5 kDa psbI gene product.

Two sites of photoinhibition of the electron transfer in oxygen evolving and Tris-treated PS II membrane fragments from spinach.

Photoinhibition was analyzed in O2-evolving and in Tris-treated PS II membrane fragments by measuring flash-induced absorption changes at 830 nm reflecting the transient P680(+) formation and oxygen evolution. Irradiation by visible light affects the PS II electron transfer at two different sites: a) photoinhibition of site I eliminates the capability to perform a 'stable' charge separation between P680(+) and QA (-) within the reaction center (RC) and b) photoinhibition of site II blocks the electron transfer from YZ to P680(+). The quantum yield of site I photoinhibition (2-3×10(-7) inhibited RC/quantum) is independent of the functional integrity of the water oxidizing system. In contrast, the quantum yield of photoinhibition at site II depends strongly on the oxygen evolution capacity. In O2-evolving samples, the quantum yield of site II photoinhibition is about 10(-7) inhibited RC/quantum. After selective elimination of the O2-evolving capacity by Tris-treatment, the quantum yield of photoinhibition at site II depends on the light intensity. At low intensity (<3 W/m(2)), the quantum yield is 10(-4) inhibited RC/quantum (about 1000 times higher than in oxygen evolving samples). Based on these results it is inferred that the dominating deleterious effect of photoinhibition cannot be ascribed to an unique target site or a single mechanism because it depends on different experimental conditions (e.g., light intensity) and the functional status of the PS II complex.

Evidence of excited state absorption in PS II membrane fragments.

Utilizing a two-beam technique in the frequency domain, the pumped absorption of PS II membrane fragments from spinach and of acetonic chlorophyll-a solutions was measured at room temperature. In a very narrow wavelength region (0.2 nm around the pump pulse wavelength) the relative test beam transmission exhibited either a decrease or an increase, respectively, dependent on the intensity of a strong pump beam. In contrast, the transmission changes of chl-a solutions were not affected by the wavelength mistuning between pump and test beam. The data obtained for PS II membrane fragments were interpreted in terms of excited state absorption of pigment-protein clusters within the light-harvesting complex of PS II. The interpretation of the small absorption band as a homogeneously broadened line led to a transversal relaxation time for chlorophyll in vivo of about 1 ps.

Studies on the electron transfer from Tyr-161 of polypeptide D-1 to P680(+) in PS II membrane fragments from spinach.

The functional connection between redox component Y z identified as Tyr-161 of polypeptide D-1 (Debus et al. 1988) and P680(+) was analyzed by measurements of laser flash induced absorption changes at 830 nm in PS II membrane fragments from spinach. It was found that neither DCMU nor the ADRY agent 2-(3-chloro-4-trifluoromethyl) anilino-3,5-dinitrothiophene (ANT 2p) affects the rate of P680(+) reduction by Y z under conditions where the catalytic site of water oxidation stays in the redox state S1. In contrast to that, a drastic retardation is observed after mild trypsin treatment at pH=6.0. This effect which is stimualted by flash illumination can be largely reversed by Ca(2+). The above mentioned data lead to the following conclusions: (a) the segment of polypeptide D-1 containing Tyr-161 and coordination sites of P680 is not allosterically affected by structural changes due to DCMU binding at the QB-site which is also located in D-1. (b) ANT 2p as a strong protonophoric uncoupler and ADRY agent does not modify the reaction coordinate of P680(+) reduction by Y z , and (c) Ca(2+) could play a functional role for the electronic and vibrational coupling between the redox groups Y z and P680. The electron transport from Y z to P680(+) is discussed within the framework of a nonadiabatic process. Based on thermodynamic considerations the reorganization energy is estimated to be in the order of 0.5 V.

Analysis of the electron transfer from Pheo- to QA in PS II membrane fragments from spinach by time resolved 325 nm absorption changes in the picosecond domain.

Absorption changes at 325 nm (delta A325) induced by 15 ps laser flashes (lambda = 650 nm) in PS II membrane fragments were measured with picosecond time-resolution. In samples with the reaction centers (RCs) kept in the open state (P I QA) the signals are characterized by a very fast rise (not resolvable by our equipment) followed by only small changes within our time window of 1.6 ns. In the closed state (PI QA-) of the reaction center the signal decays with an average half-life time of about 250 ps. It is shown that under our excitation conditions (E = 2 x 10(14) photons/cm2 per pulse) subtraction of the absorption changes in closed RCs (delta A closed 325) from those in open RCs (delta A open 325) leads to a difference signal which is dominated by the reduction kinetics of QA. From the rise kinetics of this signal and by comparison with data in the literature it is inferred that QA becomes reduced by direct electron transfer from Pheo- with a time constant of about 350 +/- 100 ps.

The involvement of lipids in light-induced charge separation in the reaction center of photosystem II.

Extraction of PS II particles with 50 mM cholate and 1 M NaCl releases several proteins (33-, 23-, 17- and 13 kDa) and lipids from the thylakoid membrane which are essential for O2 evolution, dichlorophenolindophenol (DCIP) reduction and for stable charge separation between P680(+) and QA (-). This work correlates the results on the loss of steady-state rates for O2 evolution and PS II mediated DCIP photo-reduction with flash absorption changes directly monitoring the reaction center charge separation at 830 nm due to P680(+), the chlorophyll a donor. Reconstitution of the extracted lipids to the depleted membrane restores the ability to photo-oxidize P680 reversibly and to reduce DCIP, while stimulating O2 evolution minimally. Addition of the extracted proteins of masses 33-, 23- and 17- kDa produces no further stimulation of DCIP reduction in the presence of an exogenous donor like DPC, but does enhance this rate in the absence of exogenous donors while also stimulating O2 evolution. The proteins alone in the absence of lipids have little influence on charge separation in the reaction center. Thus lipids are essential for stable charge separation within the reaction center, involving formation of P680(+) and QA (-).