Functional organization of 3D plant thylakoid membranes as seen by high resolution microscopy.

In the field of photosynthesis, only a limited number of approaches of super-resolution fluorescence microscopy can be used, as the functional architecture of the thylakoid membrane in chloroplasts is probed through the natural fluorescence of chlorophyll molecules. In this work, we have used a custom-built fluorescence microscopy method called Single Pixel Reconstruction Imaging (SPiRI) that yields a 1.4 gain in lateral and axial resolution relative to confocal fluorescence microscopy, to obtain 2D images and 3D-reconstucted volumes of isolated chloroplasts, obtained from pea (Pisum sativum), spinach (Spinacia oleracea) and Arabidopsis thaliana. In agreement with previous studies, SPiRI images exhibit larger thylakoid grana diameters when extracted from plants under low-light regimes. The three-dimensional thylakoid architecture, revealing the complete network of the thylakoid membrane in intact, non-chemically-fixed chloroplasts can be visualized from the volume reconstructions obtained at high resolution. From such reconstructions, the stromal connections between each granum can be determined and the fluorescence intensity in the stromal lamellae compared to those of neighboring grana.

Structural and spectroscopic consequences of hexacoordination of a bacteriochlorophyll cofactor in the Rhodobacter sphaeroides reaction center .

The structural and functional consequences of changing the coordination state of one of the bacteriochlorophyll (BChl) cofactors in the purple bacterial reaction center have been explored. A combination of steady state spectroscopy and X-ray crystallography was used to demonstrate that mutagenesis of residue 181 of the L-polypeptide from Phe to Arg (FL181R) causes the BChl at the accessory (B(B)) position on the so-called inactive cofactor branch to become hexacoordinated, with no significant changes to the structure of the surrounding protein. This change was accompanied by the appearance of a distinctive absorbance band at 631 nm in the room-temperature absorbance spectrum. The ligand donor was not the Arg side chain but rather an intervening water molecule, and contrary to expectations, the Mg of B(B) did not adopt a more in-plane geometry in response to hexacoordination. The mutation caused a disturbance to the detailed conformation of the BChl macrocycle that manifested in a number of subtle changes to the resonance Raman spectrum. Hexacoordination of B(B) produced a small increase in the lifetime of the excited electronic state of the primary donor bacteriochlorophylls (P*), indicating some disturbance to light-driven energy and/or electron transfer events on the time scale of a few picoseconds after light excitation. The B(B) bacteriochlorophyll returned to a pentacoordinated state in a double mutant where the FL181R mutation was combined with removal of the native axial ligand through mutation of His M182 to Leu. Experimental evidence of hexacoordinated bacteriochlorophylls in the literature on antenna proteins is considered, and possible reasons why hexacoordinated bacteriochlorophylls and chlorophylls appear to be avoided in photosynthetic proteins are discussed.

Investigation of B-branch electron transfer by femtosecond time resolved spectroscopy in a Rhodobacter sphaeroides reaction centre that lacks the Q(A) ubiquinone.

The dynamics of electron transfer in a membrane-bound Rhodobacter sphaeroides reaction centre containing a combination of four mutations were investigated by transient absorption spectroscopy. The reaction centre, named WAAH, has a mutation that causes the reaction centre to assemble without a Q(A) ubiquinone (Ala M260 to Trp), a mutation that causes the replacement of the H(A) bacteriopheophytin with a bacteriochlorophyll (Leu M214 to His) and two mutations that remove acidic groups close to the Q(B) ubiquinone (Glu L212 to Ala and Asp L213 to Ala). Previous work has shown that the Q(B) ubiquinone is reduced by electron transfer along the so-called inactive cofactor branch (B-branch) in the WAAH reaction centre (M.C. Wakeham, M.G. Goodwin, C. McKibbin, M.R. Jones, Photo-accumulation of the P(+)Q(B)(-) radical pair state in purple bacterial reaction centres that lack the Q(A) ubiquinone, FEBS Letters 540 (2003) 234-240). In the present study the dynamics of electron transfer in the membrane-bound WAAH reaction centre were studied by femtosecond transient absorption spectroscopy, and the data analysed using a compartmental model. The analysis indicates that the yield of Q(B) reduction via the B-branch is approximately 8% in the WAAH reaction centre, consistent with results from millisecond time-scale kinetic spectroscopy. Possible contributions to this yield of the constituent mutations in the WAAH reaction centre and the membrane environment of the complex are discussed.

Replacement or exclusion of the B-branch bacteriopheophytin in the purple bacterial reaction centre: the H(B) cofactor is not required for assembly or core function of the Rhodobacter sphaeroides complex.

All of the membrane-embedded cofactors of the purple bacterial reaction centre have well-defined functional or structural roles, with the exception of the bacteriopheophytin (H(B)) located approximately half-way across the membrane on the so-called inactive- or B-branch of cofactors. Sequence alignments indicate that this bacteriochlorin cofactor is a conserved feature of purple bacterial reaction centres, and a pheophytin is also found at this position in the Photosystem-II reaction centre. Possible structural or functional consequences of replacing the H(B) bacteriopheophytin by bacteriochlorophyll were investigated in the Rhodobacter sphaeroides reaction centre through mutagenesis of residue Leu L185 to His (LL185H). Results from absorbance spectroscopy indicated that the LL185H mutant assembled with a bacteriochlorophyll at the H(B) position, but this did not affect the capacity of the reaction centre to support photosynthetic growth, or change the kinetics of charge separation along the A-branch of cofactors. It was also found that mutation of residue Ala M149 to Trp (AM149W) caused the reaction centre to assemble without an H(B) bacteriochlorin, demonstrating that this cofactor is not required for correct assembly of the reaction centre. The absence of a cofactor at this position did not affect the capacity of the reaction centre to support photosynthetic growth, or the kinetics of A-branch electron transfer. A combination of X-ray crystallography and FTIR difference spectroscopy confirmed that the H(B) cofactor was absent in the AM149W mutant, and that this had not produced any significant disturbance of the adjacent ubiquinol reductase (Q(B)) site. The data are discussed with respect to possible functional roles of the H(B) bacteriopheophytin, and we conclude that the reason(s) for conservation of a bacteriopheophytin cofactor at this position in purple bacterial reaction centres are likely to be different from those underlying conservation of a pheophytin at the analogous position in Photosystem-II.

Energy transfer and trapping in the Photosystem I complex of Synechococcus PCC 7942 and in its supercomplex with IsiA.

The cyanobacterium Synechococcus PCC 7942 grown under iron starvation assembles a supercomplex consisting of a trimeric Photosystem I (PSI) complex encircled by a ring of 18 CP43' or IsiA complexes. It has previously been shown that PSI of Synechococcus PCC 7942 contains less special long-wavelength ('red') chlorophylls than PSI of most other cyanobacteria. Here we present a comparative analysis by time-resolved absorption difference and fluorescence spectroscopy of the processes of energy transfer and trapping in trimeric PSI and PSI-IsiA supercomplexes from Synechococcus PCC 7942. All experiments were performed with the primary electron donor of PSI (P700) in the oxidized state. Our data suggest that in the PSI complex the excitation energy is equilibrated with a lifetime of 0.6 ps among the so-called bulk chlorophylls, is distributed in 3-4 ps between the bulk and red chlorophylls, and is trapped in the reaction center in 19 ps. This trapping time is shorter than that observed for other cyanobacteria, which we attribute to the lower content of red chlorophylls in PSI of this organism. In the PSI-IsiA supercomplexes, the distribution of excited states is blue-shifted compared to that in PSI, leading to a lengthening of the equilibration processes. We attributed a phase of about 1 ps to initial energy equilibration steps among the IsiA and PSI core bulk chlorophylls, a 5-7 ps phase to equilibration between bulk and red chlorophylls within the PSI core, and a 38 ps phase to trapping in the reaction center. The data suggest that the excitation energy is equilibrated among the IsiA and PSI core antenna chlorophylls before trapping occurs. Data analysis based on a simple kinetic model revealed an intrinsic rate constant for energy transfer from IsiA to PSI in the range of 2+/-1 ps. Based on this value we suggest the presence of one or more linker chlorophylls between the IsiA and PSI core complexes. These results confirm that IsiA acts as an effective light-harvesting antenna for PSI.

Acquisition of photosynthetic capacity by a reaction centre that lacks the Q(A) ubiquinone; possible insights into the evolution of reaction centres?

A photosynthetically impaired strain of Rhodobacter sphaeroides containing reaction centres with an alanine to tryptophan mutation at residue 260 of the M-polypeptide (AM260W) was incubated under photosynthetic growth conditions. This incubation produced photosynthetically competent strains containing suppressor mutations that changed residue M260 to glycine or cysteine. Spectroscopic analysis demonstrated that the loss of the Q(A) ubiquinone seen in the original AM260W mutant was reversed in the suppressor mutants. In the mutant where Trp M260 was replaced by Cys, the rate of reduction of the Q(A) ubiquinone by the adjacent (H(A)) bacteriopheophytin was reduced by three-fold. The findings of the experiment are discussed in light of the X-ray crystal structures of the wild-type and AM260W reaction centres, and the possible implications for the evolution of reaction centres as bioenergetic complexes are considered.

Strong effects of an individual water molecule on the rate of light-driven charge separation in the Rhodobacter sphaeroides reaction center.

The role of a water molecule (water A) located between the primary electron donor (P) and first electron acceptor bacteriochlorophyll (B(A)) in the purple bacterial reaction center was investigated by mutation of glycine M203 to leucine (GM203L). The x-ray crystal structure of the GM203L reaction center shows that the new leucine residue packs in such a way that water A is sterically excluded from the complex, but the structure of the protein-cofactor system around the mutation site is largely undisturbed. The results of absorbance and resonance Raman spectroscopy were consistent with either the removal of a hydrogen bond interaction between water A and the keto carbonyl group of B(A) or a change in the local electrostatic environment of this carbonyl group. Similarities in the spectroscopic properties and x-ray crystal structures of reaction centers with leucine and aspartic acid mutations at the M203 position suggested that the effects of a glycine to aspartic acid substitution at the M203 position can also be explained by steric exclusion of water A. In the GM203L mutant, loss of water A was accompanied by an approximately 8-fold slowing of the rate of decay of the primary donor excited state, indicating that the presence of water A is important for optimization of the rate of primary electron transfer. Possible functions of this water molecule are discussed, including a switching role in which the redox potential of the B(A) acceptor is rapidly modulated in response to oxidation of the primary electron donor.

Oxidation of the two beta-carotene molecules in the photosystem II reaction center.

We present a spectroscopic characterization of the two nonequivalent beta-carotene molecules in the photosystem II reaction center. Their electronic and vibrational properties exhibit significant differences, reflecting a somewhat different configuration for these two cofactors. Both carotenoid molecules are redox-active and can be oxidized by illumination of the reaction centers in the presence of an electron acceptor. The radical cation species show similar differences in their spectroscopic properties. The results are discussed in terms of the structure and unusual function of these carotenoids. In addition, the attribution of resonance Raman spectra of photosystem II preparations excited in the range 800-900 nm is discussed. Although contributions of chlorophyll cations cannot be formally ruled out, our results demonstrate that these spectra mainly arise from the cation radical species of the two carotenoids present in photosystem II reaction centers.