Antenna Structures and Core Complexes of A.marina

As an example of an exotic cyanobacterium that has been analysed and modelled in detail with special attention given to EET processes in its antenna system and subsequent ET we use the cyanobacterium A.marina. The A.marina is an exception because it contains mainly Chl d in the membrane intrinsic antenna and RC complexes. The Qy band of Chl d (Qy,(0,0)) is red-shifted to about 699 nm in ethanol solution, as shown in Figure 36, compared to the absorption maximum of Chl a at 675 nm (see Figure 21). The absorption maximum of the Chl d antenna in A.marina was found red-shifted by another 20 nm to an absorption maximum at 719 nm for the Qy(0,0) transition according to our measurements (see Figure 34) while according to (Miyashita et al., 1997) the Chl d absorp?tion maximum appears at 714 nm in A.marina. Former measurements showed a peak at 717 nm for the Qy band of Chl d in living cells of A.marina (Schmitt, 2011). This red shift most probably influenced by the surrounding protein matrix.

Absorption (without correction of scattering background) of whole cells of A.marina (upper panel) and corrected spectrum with second derivative (lower panel)

Figure 34. Absorption (without correction of scattering background) of whole cells of A.marina (upper panel) and corrected spectrum with second derivative (lower panel).

The spectrum shown in Figure 34, upper panel, is not corrected for scattered light. The whole cells with a diameter of about 1 pm exhibit strong scattering; this leads to an increase of the background level at shorter wavelengths. Figure 34 also shows the absorption of the PBP antenna in A.marina between 610 nm and 650 nm (see chap. 2.4.4.) and the Bx and By absorption band of Chl d which exhibits highest absorption at 468 nm in living cells (see Figure 34 in comparison to Figure 36 for the calculated Chl d spectrum).

Figure 35 shows the energy levels and selected electronic transitions, including vibrational states of Chl d, in 40:1 methanol: acetonitrile at 170 K according to (Nieuwenburg, 2003). In (Nieuwenburg, 2003) the dipole strengths and spectral linewidths of the different transitions are published as shown in Figure 35. This information was used to plot the relative contributions of these transitions to the absorption spectrum (see Figure 36, for further details see Schmitt, 2011).

Energetic states and characteristic transitions of Chl d in 40:1 methanol

Figure 35. Energetic states and characteristic transitions of Chl d in 40:1 methanol: acetonitrile at 170 K according to (Nieuwenburg, 2003).

The broad absorption spectrum of pigments caused by intramolecular vibrational states is of importance for achieving an efficient solar energy exploitation and fulfilling the resonance condition between the fluorescence spectrum of donor pigments and the absorption of acceptor pigments. The latter is necessary for an efficient energy transfer along the antenna pigments according to the FRET-mechanism.

The thylakoid membrane in A.marina is ring-shaped and contains densely packed PBP antenna rods. A.marina has a uniquely composed light-harvesting system, as schematically shown in Figure 37 (right side). The PBP antenna has a simpler rod shaped structure than that of PBS in typical cyanobacteria (Schmitt, 2011).

Calculated absorption spectrum of Chl d using the transitions shown in Figure 35 and values for the dipole strengths, spectral linewidths and extinction maximum at 699 nm

Figure 36. Calculated absorption spectrum of Chl d using the transitions shown in Figure 35 and values for the dipole strengths, spectral linewidths and extinction maximum at 699 nm.

Overall geometry of PBS of typical cyanobacteria

Figure 37. Overall geometry of PBS of typical cyanobacteria (left side) and rod shaped PBP antenna structure of A.marina (right side) according to (Marquardt et al., 1997), Both PBP contain PC and APC. The PBS additionally contains PE trimers.

This work confirms the PBP structure suggested by (Marquardt et al., 1997), (Figure 37) through transmission electron microscopy of the PBP antenna complexes of A.marina as shown in Figure 38. In comparison to the pictures presented in (Marquardt et al., 1997), the PBP complexes appear slightly larger with extensions of up to 40 nm in length and 20 nm in diameter. The contrast of the protein structure in the electron beam is low. Therefore a procedure of negative staining had to be performed to achieve a picture of the PBP structure.

Electron microscopic study of PBP preparations o

Figure 38. Electron microscopic study of PBP preparations of A.marina in buffer containing phosphate after negative staining with Na4[W12SiO40]. The PBP antenna complexes appear transparent (white) due to the process of negative staining while the staining salt leads to a dark green contrast.

A carbon film on a copper net acts as a sample holder in transmission electron microscopic studies and was incubated into a concentrated PBP solution in phosphate buffer for 24 hours. Then the carbon film was put into a 4% (w/w) solution of Sodium-Silicotungstate (Na4[W12SiO40]) which contains the heavy metal tungsten (W) which has a high scattering cross section for keV electrons. The specimen is left in the Na4[W12SiO40] solution for about 30 min. After this negative staining procedure, the sample is washed for circa two minutes in distilled water and left to dry for 24 hours. The carbon film can be directly used in the electron microscope.

Typical absorption spectra of PBP antenna complexes of A.marina dissolved in aqueous phosphate containing buffer are shown in Figure 39 (black line). For the purpose of comparison, the absorption of isolated PC trimers (green curve) and isolated APC trimers (red curve) are shown in Figure 29. The large absorption gap of Chl d between 500 nm and 650 nm (see Figure 36) is partially filled by the absorption of the PBP antenna as shown in Figure 39.

Interestingly, the absorption spectrum of isolated PBP antenna complexes from A.marina (black curve in Figure 39) is less broadened than the spectrum of isolated PC trimers (green curve in Figure 39). Since the PC trimers couple to hexamers, one would expect a slight broadening of the PBP absorption compared to isolated PC trimers. In addition, the PBP antenna of A.marina contains APC which should contribute to the red edge of the spectrum at 653 nm (see Figure 29 and red line in Figure 39).

Absorption spectrum of PBP isolated from the cyanobacteriu

Figure 39. Absorption spectrum of PBP isolated from the cyanobacterium A.marina (black curve) in comparison to isolated PC trimers (green line) and isolated APC trimers (red line) as shown in Figure 29 according to (Theiss, 2006). The trimers were diluted in buffered aqueous solution. Image reproduced with permission.

The narrow PBP spectrum (black curve) could not be explained fully. However, it seems that there is some absorption at wavelengths >680 nm. These long wavelength absorption states play an important role for the energy transfer between the PBP antenna and Chl d (see chap. 2.6)

The PBPs of A.marina have been reported to consist of four hexameric units (Marquardt et al., 1997) (see Figure 37, right side). Each homo- hexamer covalently binds 18 PCB molecules as chromophores. The PC/APC heterohexamer is found to contain 9 PCB and 6 APCB chromo- phores (see Figure 28). Isolated PBPs from A.marina exhibit a fluorescence maximum at 665 nm (APC) with a shoulder at about 655 nm (PC) at room temperature (Marquardt et al., 1997).

 
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