Phycobilisomes in Cyanobacteria
The antenna complexes of photosynthetic organisms enhance and regulate the effective absorption cross-section by connecting a huge number of pigments to the RC and by regulating the connectivity and excited state lifetime. The modulation by protein environment and the coupling of pigments determines the probability distribution of EET pathways. Additionally, the high number of nondegenerated states of all pigments in these antenna complexes enables light absorption in a wider spectral range than single pigment molecules. In photosynthetic organisms different molecules are bound to peripheral antenna complexes which close the absorption gap of the chlorophyll molecules in the green spectral range and therefore raise the efficiency of the antenna. Such pigments are carotenoids in LHCII (see chap. 2.4.1) and phycobiliprotein (PBP) complexes containing phycoerythrin (PE), phycocyanin (PC) and allophyco- cyanin (APC) in cyanobacteria. The pigments are open porphyrin ring systems exhibiting strong absorption in the green and yellow spectral range, thus being responsible for the blue color of cyanobacteria.
Figure 28, left side, shows the structure of C-PC of typical cyanobacteria like Synechocystis and Synechococcus. Each monomeric protein subunit contains three phycocyanobilin (PCB) chromophores (9 pigments in one trimer). In Figure 28, right side, the APC trimer is shown. This trimeric protein binds 6 pigments (two chromophores per monomer).
The PC monomers consist of the a-subunit containing the в-84 chro- mophore and the в-subunit containing two chromophores, the в-84 PCB and the a-155 PCB (see Figure 28, left side). The APC monomers contain one PCB molecule in the a-subunit (a-80) and one pigment in the в-sub- unit (в-81) (see Figure 28, right side).
Figure 28. Three monomeric phycocyanobilin containing PCs (left side) and the tetrameric structure of APC (right side). The PC structure was generated with a protein database using the data published in (Nield et al., 2003) and graphically improved with Corel Draw®. The APC structure was generated from the data published in (Murray et al., 2007) improved with Corel Draw®.
Figure 29. Absorption spectra of isolated PC trimers (green line) and isolated APC trimers (red line) in buffered aqueous solution (data redrawn from Theiss et al., 2011). Image reproduced with permission.
The absorption spectra of PC and APC trimers exhibit characteristic bands that result from strong excitonic coupling of the pigments. The small shoulder in the PC absorption spectrum (green curve in Figure 29) at 650 nm most probably represents the absorption of the lowest excitonic state. This state is strongly visible in the APC spectrum shown in Figure 29 (red curve) at 653 nm. In APC trimers (see Figure 28, right side) there exists a well defined excitonically coupled dimer containing the two PCB chromophores a-80 and в-81 of different monomers while in the PC trimers all three pigments are rather weakly excitonically coupled between different monomers with the a-84 chromophore in the a-subunit of one monomer and the в-84 PC and the в-155 PC in the в-subunit of another monomer (see Figure 30, right side). Sauer and Scheer (1988) calculated that the strongest coupling with a value of 56 cm-1 exists between a-84 and в-84 of different subunits. The exact structural constitution of the pigments in the PC and APC trimers is of high relevance for the shape of the absorption spectrum due to the formation of strongly absorbing excitonic states. The coupling makes is impossible to extract the optical properties of an isolated pigment from the absorption spectrum of the pigment- protein complex (e.g. a PC monomer). This is shown in (Debreczeny et al., 1993). To analyse the properties of individual compounds, the sample has to be decomposed into the individual constituents. This leads to a loss of information on the coupling of the pigments, however, since this coupling is an essential physical property determining the functionality of the structure, it is not possible to analyse the function of the whole complex by studying the decomposed compartments. The functionality is directly connected with the whole structure. This is in some way related to the fact that a whole structure cannot be explained by its (isolated) parts, as was mentioned at the outset of this book (Heisenberg, 1986).
Figure 30. Structure of monomeric PC (left side) generated with protein data and graphically improved with Corel Draw®. The protein monomer consists of two subunits: The в-subunit shown in green binding the two chromophores в-84 and в-155 and the a-subunit (shown in blue) which binds only one chromophore, the a-84 PCB. The absorption maxima and distances between the different PCB molecules in the trimeric PC calculated by (Sauer and Scheer, 1988) and (Suter and Holzwarth, 1987; Holzwarth, 1991) are shown on the right side. Image reproduced with permission.
The effect of excitonic coupling of PCB molecules in different protein subunits can be analysed if the absorption of PC monomers is compared with PC trimers as shown by Debreczeny et al. (1995a, 1995b). An accurate comparison revealed that the small shoulder at 650 nm in the spectrum of isolated PC trimers (see Figure 29) is the result of a coupling between a-84 and ^-84 in different monomers. With high resolved fs-absorption spectroscopy Sharkov et al. showed that, in APC trimers, excitonic relaxation in the coupled a-80/ в-81 dimer between different monomers occurs with 440 fs (Sharkov et al., 1992, 1996). Much slower excitation energy transfer processes, with time constants of 140 ps, were observed between the different chromophores in one monomeric subunit. The coupling strength of the different chromophores in PC mono?mers and trimers was analysed by (Sauer and Scheer, 1988). The absorption wavelengths and calculated distances of the PC molecules inside the trimeric PCB containing protein disc are shown in Figure 30.
The strongest interaction between pigment molecule and protein occurs via a sulfur bridge between the pyrrole ring of the PCB pigment a-84 and a cysteine group of the protein as shown in Figure 31. Hydrogen bonds help to stabilize the structure and in-plane geometry of the chro- mophore molecules and the protein.
Figure 31. PCB chromophore a-84 bound to the protein matrix via a sulfur bridge between PCB and the cysteine of the protein. (Figure 31 was generated with ChemSketch V®).
In addition to the sulfur bridge shown in Figure 31, there are several strong hydrophobic interactions between pigments and protein structure. Such attractive van-der-Waals forces decay with the sixth power of the distance between pigment and protein. Therefore only the strongest interactions determine the structure.
Figure 32 shows a view on the ^-84 PCB chromophore found in the monomeric PC structure that interacts with leucine along the shortest hydrophobic interaction. From this illustration one can get an idea of the flexibility of the protein matrix comparable to an ensemble of coupled springs. This spring like structural arrangement results in a broad distribution of oscillation modes that efficiently dissipates electronic excess energy in the pigments and modulates the electronic properties of the pigments bound to the protein quasistatically and dynamically. The quasistatic modulation leads to a broad Gaussian distribution of chromophore site energies while the dynamic modulation leads to a homogeneous broadening of the chromophore ensemble and even is assumed to be involved in dynamic excited state localisation effects that supports the EET.
Figure 32. в-84 chromophore in PC bound to the protein в-subunit. The shortest hydrophobic interaction (3.2 °A) between the chromophore and the protein
is shown as green dashed line. The figure was generated with protein data base
according to the data published in (Nield et al., 2003) and graphically improved with Corel Draw®.
The PC and APC trimers (see Figure 28) tend to aggregate further in the so-called “face-to-face“ dimerization which forms hexameric structures (for further detail see e.g. Theiss, 2006; Holzwarth, 1991; Glazer, 1985). These hexamers undergo further aggregation to form so-called PBP antenna rods, which in their turn aggregate to huge (up to 50 nm in diameter) phycobilisomes (PBS) as shown in Figure 33 and Figure 37 (left side). In Figure 37, the APC-containing hexameric structures are present only in the form of single discs, but they are also known to form rodshaped staples of hexameric discs which “lie” on the outer thylakoid membrane. Some cyanobacterial PBS additionally contain phycoerythrin (PE) as well as PC and APC hexamers in the PBS.
The properties of PBS are well understood and therefore PBS is quite a good system for reference measurements of energy transfer. In the study presented here measurements of PBS from the cyanobacterium Synecho- cystis PCC 6803, containing PC and APC as schematically shown in Figure 37, were performed. It was found that energy migrates in about 200 ps from PC to Chl a in Synechocystis which is a typical value for PBS of common cyanobacteria in agreement with (Holzwarth, 1991; Trissl, 2003; Glazer, 1985).
Figure 33. Schematic view of the association of PBS with the PS II inside the thylakoid membrane according to (Hader, 1999). Image reproduced with permission.
It is assumed that the PBS are mainly associated with the PS II core complex of the thylakoid membrane (see Figure 33). Most cyanobacteria contain high amounts of PBS; this makes the PBS chromophores the most important light-harvesting pigments found in cyanobacteria. PBS undergo state transitions from the state indicated in Figure 33, where the PBS are associated with the PS II, and a state where they are associated with PS I. Other studies show that similar behaviour is also approved for the LHCII in higher plants (Minagawa, 2010). Figure 33 indicates the electron pathways after light absorption. Details for the electron transfer chain are found in chap. 1.4.