Intramolecular light induced activation of a Salen-Mn(III) complex by a ruthenium photosensitizer.

We have designed a molecular system consisting of a heteroleptic [Ru(bpy)(2)L](2+) chromophore covalently linked to a Mn(III)-Salen unit. We demonstrate the light induced oxidation of the Mn(III) center in this putative photo-catalyst assembly to a Mn(IV) high spin intermediate. Both oxidation states have been characterized by transient absorption and EPR techniques.

D1 protein variants in Photosystem II from Thermosynechococcus elongatus studied by low temperature optical spectroscopy.

In Photosystem II (PSII) from Thermosynechococcus elongatus, high-light intensity growth conditions induce the preferential expression of the psbA(3) gene over the psbA(1) gene. These genes encode for the D1 protein variants labeled D1:3 and D1:1, respectively. We have compared steady state absorption and photo-induced difference spectra at <10 K of PSII containing either D1:1 or D1:3. The following differences were observed. (i) The pheophytin Q(x) band was red-shifted in D1:3 (547.3 nm) compared to D1:1 (544.3 nm). (ii) The electrochromism on the Pheo(D1) Q(x) band induced by Q(A)(-) (the C550 shift) was more asymmetric in D1:3. (iii) The two variants differed in their responses to excitation with far red (704 nm) light. When green light was used there was little difference between the two variants. With far red light the stable (t(1/2)>50 ms) Q(A)(-) yield was approximately 95% in D1:3, and approximately 60% in D1:1, relative to green light excitation. (iv) For the D1:1 variant, the quantum efficiency of photo-induced oxidation of side-pathway donors was lower. These effects can be correlated with amino acid changes between the two D1 variants. The effects on the pheophytin Q(x) band can be attributed to the hydrogen bond from Glu130 in D1:3 to the 13(1)-keto of Pheo(D1), which is absent for Gln130 in D1:1. The reduced yield with red light in the D1:1 variant could be associated with either the Glu130Gln change, and/or the four changes near the binding site of P(D1), in particular Ser153Ala. Photo-induced Q(A)(-) formation with far red light is assigned to the direct optical excitation of a weakly absorbing charge transfer state of the reaction centre. We suggest that this state is blue-shifted in the D1:1 variant. A reduced efficiency for the oxidation of side-pathway donors in the D1:1 variant could be explained by a variation in the location and/or redox potential of P+.

Identification of the QY excitation of the primary electron acceptor of photosystem II: CD determination of its coupling environment.

Low-temperature absorption and CD spectra, measured simultaneously, are reported from Photosystem II (PS II) reduced with sodium dithionite. Spectra were obtained using PS II core complexes before and after photoaccumulation of Pheo(D1)(-), the anion of the primary acceptor. For plant PS II, Pheo(D1)(-) was generated under conditions in which the primary plastoquinone was present as an anion (Q(A)(-)) and as a modified species taken to be the neutral doubly reduced hydroquinone (Q(A)H(2)). The bleaches observed upon Pheo(D1)(-) formation in the presence of Q(A)(-) are shifted to the blue compared those in the presence of Q(A)H(2). This is attributed to the influence of the charge on Q(A)(-), and this effect mirrors the well-known electrochromic effect of Q(A)(-) on the neutral pigments. The absorption bleaches induced by Pheo(D1) reduction are species dependent. Structured changes of the CD in the 680-690 nm spectral region are seen upon photoaccumulation of Pheo(D1)(-) in PS II from plant, Synechocystis and Thermosynechococcus vulcanus. These CD changes are shown to be consistent with the overall electronic assignments of Raszewski et al. [Raszewski et al. Biophys. J. 2008, 95, 105], which place the dominant Pheo(D1) excitation near 672 nm. CD changes associated with Pheo(D1) reduction are modeled to arise from the shift and intensity changes of two CD features: one predominately of Chl(D1) character, the other predominately Pheo(D2) in character. The assignments are also shown to account for the Q(Y) absorption changes in samples where the quinone is its charged (Q(A)(-)) and neutral (Q(A)H(2)) states.

The S(1) split signal of photosystem II; a tyrosine-manganese coupled interaction.

Detailed optical and EPR analyses of states induced in dark-adapted PS II membranes by cryogenic illumination permit characterization and quantification of all pigment derived donors and acceptors, as well as optically silent (in the visible, near infrared) species which are EPR active. Near complete turnover formation of Q(A)((-)) is seen in all centers, but with variable efficiency, depending on the donor species. In minimally detergent-exposed PS II membranes, negligible (<5%) oxidation of chlorophyll or carotenoid centers occurs for illumination temperatures 5-20 K. An optically silent electron donor to P680(+) is observed with the same decay kinetics as the S(1) split signal. Cryogenic donors to P680(+) seen are: (i) transient (t(1/2) approximately 150 s) tyrosine related species, including 'split signals' ( approximately 15% total centers), (ii) reduced cytochrome b(559) ( approximately 30-50% centers), and (iii) an organic donor, possibly an amino acid side chain, ( approximately 30% centers).

Quantum efficiency distributions of photo-induced side-pathway donor oxidation at cryogenic temperature in photosystem II.

We monitored illuminated-minus-dark absorption difference spectra in the range of 450-1100 nm induced by continuous illumination at 8 K of photosystem II (PSII) core complexes from Thermosynechococcus elongatus. The photo-induced oxidation of the side-path donors Cytb(559), beta-carotene and chlorophyll Z, as well as the concomitant stable (t(1/2) > 1 s) reduction of the first plastoquinone electron acceptor, Q(A) (monitored by the well-known 'C550' shift), were quantified as a function of the absorbed photons per PSII. The Q(A) photo-induced reduction data can be described by three distinct quantum efficiency distributions: (i) a very high efficiency of approximately 0.5-1, (ii) a middle efficiency with a very large range of approximately 0.014-0.2, and (iii) a low efficiency of approximately 0.002. Each of the observed side-path donors exhibited similar quantum efficiency distributions, which supports a branched pathway model for side-path oxidation where beta-carotene is the immediate electron donor to the photo-oxidized chlorophyll (P680(+)). The yields of the observed side-path donors account quantitatively for the wide middle efficiency range of photo-induced Q(A) reduction, but not for the PSII fractions that exhibit the highest and lowest efficiencies. The high-efficiency component may be due to Tyr(Z) oxidation. A donor that does not exhibit an identified absorption in the visible-near-IR region is mainly responsible for the lowest efficiency component.

Photophysical behavior and assignment of the low-energy chlorophyll states in the CP43 proximal antenna protein of higher plant photosystem II.

We have employed absorption, circular dichroism (CD), and persistent spectral hole-burning measurements at 1.7 K to study the photoconversion properties and exciton coupling of low-energy chlorophylls (Chls) in the CP43 proximal antenna light-harvesting subunit of photosystem II (PSII) isolated from spinach. These approximately 683 nm states act as traps for excitation energy in isolated CP43. They "bleach" at 683 nm upon illumination and photoconvert to a form absorbing in the range approximately 660-680 nm. We present new data that show the changes in the CD spectrum due to the photoconversion process. These changes occur in parallel with those in absorption, providing evidence that the feature undergoing the apparent bleach is a component of a weakly exciton-coupled system. From our photoconversion difference spectra, we assign four states in the Chl long-wavelength region of CP43, two of which are the known trap states and are both highly localized on single Chls. The other two states are associated with weak exciton coupling (maximally approximately 50 cm(-)(1)) to one of these traps. We propose a mechanism for photoconversion that involves Chl-protein hydrogen bonding. New hole-burning data are presented that indicate this mechanism is distinct to that for narrow-band spectral hole burning in CP43. We discuss the photophysical behavior of the Chl trap states in isolated CP43 compared to their behavior in intact PSII preparations. The latter represent a more intact, physiological complex, and we find no clear evidence that they exhibit the photoconversion process reported here.

Charge separation in photosystem II core complexes induced by 690-730 nm excitation at 1.7 K.

The illumination of oxygen-evolving PSII core complexes at very low temperatures in spectral regions not expected to excite P680 leads to charge separation in a majority of centers. The fraction of centers photoconverted as a function of the number of absorbed photons per PSII core is determined by quantification of electrochromic shifts on Pheo(D1). These shifts arise from the formation of metastable plastoquinone anion (Q(A)(-)) configurations. Spectra of concentrated samples identify absorption in the 700-730 nm range. This is well beyond absorption attributable to CP47. Spectra in the 690-730 nm region can be described by the 'trap' CP47 absorption at 689 nm, with dipole strength of approximately 1 chlorophyll a (chl a), partially overlapping a broader feature near 705 nm with a dipole strength of approximately 0.15 chl a. This absorption strength in the 700-730 nm region falls by 40% in the photoconverted configuration. Quantum efficiencies of photoconversion following illumination in the 690-700 nm region are similar to those obtained with green illumination but fall significantly in the 700-730 nm range. Two possible assignments of the long-wavelength absorption are considered. Firstly, as a low intensity component of strongly exciton-coupled reaction center chlorin excitations and secondly as a nominally 'dark' charge-transfer excitation of the 'special pair' P(D1)-P(D2). The opportunities offered by these observations towards the understanding of the nature of P680 and PSII fluorescence are discussed.

Magneto-optic spectroscopy of a protein tetramer binding two exciton-coupled chlorophylls.

In vitro chlorophyll (Chl) aggregates have often served as models for in vivo forms of long-wavelength Chl. However, the interaction of protein-bound Chl molecules is typically different than that occurring in solvent-based self-aggregates. We have chosen a water-soluble Chl-binding protein (WSCP) from cauliflower in order to help characterize the spectroscopic properties of Chl in a single well-defined native environment and also to study the pigment-pigment (exciton) interactions present in assemblies of this protein. WSCP forms tetrameric units upon binding two Chl molecules. We present the absorption, circular dichroism (CD), magnetic circular dichroism (MCD), and emission spectra at 1.7 K of recombinant WSCP tetramers containing either Chl a or Chl d. The spectroscopic characteristics provide evidence for significant exciton interaction between equivalent Chl molecules. Our simple exciton analysis allows an estimate of the molecular geometry of the dimer, which is predicted to have an "open sandwich"-type structure. We find that the ratio of the magnetic circular dichroism to absorption, deltaA/A, is substantially increased (approximately 60%) for Chl a in this system compared to its value in solution. This increase is in marked contrast to substantial reductions (>50%) of deltaA/A seen in solvent-based Chl aggregates and in photosynthetic reaction centers. Current theoretical models are unable to account for such large variations in the MCD to absorption ratio for Chl. We propose that spectroscopic studies of WSCP mutants will enable a fundamental understanding of Chl-Chl and Chl-protein interactions.

Spectroscopic studies of photosystem II in chlorophyll d-containing Acaryochloris marina.

Photosystem II (PSII) electron transfer (ET) in the chlorophyll d-containing cyanobacterium Acaryochloris marina (A. marina) was studied by time-resolved electron paramagnetic resonance (EPR) spectroscopy at room temperature, chlorophyll fluorescence, and low-temperature optical spectroscopy. To maximize the ability to measure PSII ET in the intact cells of this organism, growth conditions were optimized to provide the highest specific O(2) activity and the instrumental parameters for the EPR measurements of tyrosine Z (Y(Z)) reduction were adjusted to give the best signal-to-noise over spectral resolution. Analysis of the Y(Z)(*) reduction kinetics revealed that ET to the oxygen-evolving complex on the donor side of PSII in A. marina is indistinguishable from that in higher plants and other cyanobacteria. Likewise, the charge recombination kinetics between the first plastoquinone acceptor Q(A) and the donor side of PSII monitored by the chlorophyll fluorescence decay on the seconds time scale are not significantly different between A. marina and non-chlorophyll d organisms, while low-temperature optical absorption spectroscopy identified the primary electron acceptor in A. marina as pheophytin a. The results indicate that, if the PSII primary electron donor in A. marina is made up of chlorophyll d instead of chlorophyll a, then there must be very different interactions with the protein environment to account for the ET properties, which are similar to higher plants and other cyanobacteria. Nevertheless, the water oxidation mechanism in A. marina is kinetically unaltered.

Oxygen-evolving Photosystem II core complexes: a new paradigm based on the spectral identification of the charge-separating state, the primary acceptor and assignment of low-temperature fluorescence.

We review our recent low-temperature absorption, circular dichroism (CD), magnetic CD (MCD), fluorescence and laser-selective measurements of oxygen-evolving Photosystem II (PSII) core complexes and their constituent CP 4 3, CP 47 and D1/D2/cytb(559) sub-assemblies. Quantitative comparisons reveal that neither absorption nor fluorescence spectra of core complexes are simple additive combinations of the spectra of the sub-assemblies. The absorption spectrum of the D1/D2/cytb(559) component embedded within the core complex appears significantly better structured and red-shifted compared to that of the isolated sub-assembly. A characteristic MCD reduction or 'deficit' is a useful signature for the central chlorins in the reaction centre. We note a congruence of the MCD deficit spectra of the isolated D1/D2/cytb(559) sub-assemblies to their laser-induced transient bleaches associated with P 680. A comparison of spectra of core complexes prepared from different organisms helps distinguish features due to inner light-harvesting assemblies and the central reaction-centre chlorins. Electrochromic spectral shifts in core complexes that occur following low-temperature illumination of active core complexes arise from efficient charge separation and subsequent plastoquinone anion (Q(A)(-)) formation. Such measurements allow determinations of both charge-separation efficiencies and spectral characteristics of the primary acceptor, Pheo(D1). Efficient charge separation occurs with excitation wavelengths as long as 700 nm despite the illuminations being performed at 1.7 K and with an extremely low level of incident power density. A weak, homogeneously broadened, charge-separating state of PSII lies obscured beneath the CP 47 state centered at 690 nm. We present new data in the 690-760 nm region, clearly identifying a band extending to 730 nm. Active core complexes show remarkably strong persistent spectral hole-burning activity in spectral regions attributable to CP 43 and CP 47. Measurements of homogeneous hole-widths have established that, at low temperatures, excitation transfer from these inner light-harvesting assemblies to the reaction centre occurs with approximately 70-270 ps(-1) rates, when the quinone acceptor is reduced. The rate is slower for lower-energy sub-populations of an inhomogeneously broadened antenna (trap) pigment. The complex low-temperature fluorescence behaviour seen in PSII is explicable in terms of slow excitation transfer from traps to the weak low-energy charge-separating state and transfer to the more intense reaction-centre excitations near 685 nm. The nature and origin of the charge-separating state in oxygen-evolving PSII preparations is briefly discussed.

Probing the lowest energy chlorophyll a states of photosystem II via selective spectroscopy: new insights on P680.

We present the wavelength dependence of homogeneous holewidths of persistent spectral holes burnt in O2-evolving Photosystem II core complexes isolated from spinach, in the temperature range 2.5-8 K. The data supports the assignment that those chlorophylls which undergo persistent spectral hole-burning are specific CP43 and CP47-trap states that transfer their excitation energy to the reaction center. The lifetime-limited holewidths show that when PS II is in the S1(QA -) (closed) state, the CP43/CP47-trap states have excited-state lifetimes in the range from 70 to 270 ps. These lifetimes correspond to excitation transfer rates to the reaction center, and are far slower than required for models in which the PS II reaction center (P680) acts as a 'shallow-trap' for excitations. For wavelengths at which both traps absorb, the hole shape is clearly a composite of two Lorentzians, corresponding to hole-burning in both states simultaneously. The temperature dependence of the homogeneous holewidth does not follow the usual T1.3 dependence found in many chlorophyll-protein systems. Our data indicates T 2 temperature dependence, typically found in crystalline systems where the chromophore is coupled to localized phonon modes.

Assignment of the low-temperature fluorescence in oxygen-evolving photosystem II.

Low-temperature absorption and fluorescence spectra of fully active cores and membrane-bound PS II preparations are compared. Detailed temperature dependence of fluorescence spectra between 5 and 70 K are presented as well as 1.7-K fluorescence line-narrowed (FLN) spectra of cores, confirming that PS II emission is composite. Spectra are compared to those reported for LHCII, CP43, CP47 and D1/D2/cytit b559 subunits of PS II. A combination of subunit spectra cannot account for emission of active PS II. The complex temperature dependence of PS II fluorescence is interpretable by noting that excitation transfer from CP43 and CP47 to the reaction centre is slow, and strongly dependent on the precise energy at which a 'slow-transfer' pigment in CP43 or CP47 is located within its inhomogeneous distribution. PS II fluorescence arises from CP43 and CP47 'slow-transfer' states, convolved by this dependence. At higher temperatures, thermally activated excitation transfer to the PS II charge-separating system bypasses such bottlenecks. As the charge-separating state of active PS II absorbs at >700 nm, PS II emission in the 680-700 nm region is unlikely to arise from reaction centre pigments. PS II emission at physiological temperatures is discussed in terms of these results.

Multiple transient spectral holes in the R1-line of NaMgAl(oxalate)(3).9H2O:Cr(III). Probing excited and ground state g-factors in an external magnetic field of 15 mT.

The gz-factors of the 4A2 ground state and the 2E excited state of chromium(III) doped into NaMgAl(oxalate)(3).9H2O are accurately determined at 2.5 K by the observation of multiple transient spectral holes in the R1-line (2E<--4A2 transition) in minute magnetic fields of approximately 15 mT.