Analysis of Some Optical Properties of a Native and Reconstituted Photosystem II Antenna Complex, CP29:  Pigment Binding Sites Can Be Occupied by Chlorophyll a or Chlorophyll b and Determine Spectral Forms

Abstract

The minor photosystem II antenna complex CP29(Lhcb-4) has been reconstituted in vitro with the Lhcb-4 apoprotein, overexpressed in Escherichia coli, and the native pigments. Modulation of the pigment composition during reconstitution yields binding products with markedly different chlorophyll a/b binding ratios even though the total number of bound chlorophylls (a plus b) remains constant at eight. A thermodynamic analysis of steady state absorption and fluorescence spectra demonstrates that all chlorophylls are energetically coupled, while the kinetics of chlorophyll photooxidation indicate that triplet chlorophyll-carotenoid coupling is also conserved during pigment binding in vitro. The influence of the chlorophyll a/b binding ratio on the absorption spectra measured at 72 and 300 K is analyzed for the Qy absorption region. Increased chlorophyll b binding leads to large increases in absorption in the 640-660 nm region, while absorption in the 675-690 nm interval decreases markedly. These changes are analyzed in terms of a Gaussian decomposition description in which the eight subbands display a temperature-dependent broadening in agreement with the weak electron-phonon coupling demonstrated for other antenna chlorophyll spectral forms. In this way, we demonstrate that increased chlorophyll b binding leads to increased absorption intensity associated with the subbands at 640, 648, 655, and 660 nm and decreased intensity for the long wavelength subbands at 678 and 684 nm. The wavelength position of all subbands is unchanged. The above data are interpreted to indicate that CP29 has eight chlorophyll binding sites, many or all of which can be occupied by either chlorophyll a or chlorophyll b according to the conditions in which pigment binding occurs. Chlorophyll b absorption is primarily associated with four subbands located at 640, 648, 655, and 660 nm. The invariance of the wavelength position of the absorption bands in recombinant products with different chlorophyll a/b binding stoichiometries is discussed in terms of the mechanism involved in the formation of spectral bands. We conclude that pigment-protein interactions dominate in the determination of spectral heterogeneity with probably only minor effects on absorption associated with pigment-pigment interactions.

Orientation of Chlorophyll Transition Moments in the Higher-Plant Light-Harvesting Complex CP29

“…Such a result suggests that the removal of one pigment from the complex did not introduce major changes in the spectral properties of the remaining chromophores. This is in agreement with previous work in which the effect of the protein binding site was shown to be the major determinant of pigment absorption (20,21). A representative example of a WT minus mutant Abs spectrum is shown in Figure 4 (solid line) for the mutant H216F.…”

“…The transition wavelength and orientation parameters that were used for the spectral reconstruction are reported in Table 3. The assignment of the optical transition to each Chl binding site in antenna systems is still an open question which was widely studied by optical spectroscopy and Gaussian decomposition either in the case of CP29 (20) or more generally for the Lhc family in PSII (5,16). The single-site mutational approach coupled to the spectroscopic analysis is a powerful tool in pinpointing the contribution of missing Chl in the differential spectra (WT vs mutants, see panels A and B of Figure 3).…”

Carotenoid-binding Sites of the Major Light-harvesting Complex II of Higher Plants

“…4 (A and B). In the 630 -660 nm range, corresponding to Chl b absorption (19,21), LHCII lutein showed a strong decrease in the amplitude of the 652 nm peak, whereas the 640 nm absorption component was slightly increased. Differences were also observed in the Chl a absorption region (660 -684 nm), where the amplitude of the 677 nm transition was decreased, whereas higher absorption was observed at 663 nm.…”

“…The pigment composition of the recombinant proteins was determined by a combined approach of HPLC analysis and fitting of the acetone extract spectrum with the sum of spectra of purified pigments (3). The pigment/protein stoichiometry was also determined as described previously (20,21). The results are reported in Table I.…”

“…Further insight into the photoprotection function is given by evaluation of the y 0 value and the bleaching times separately for Chl a and Chl b. The area from 630 nm to the isosbestic point at 663 nm is due to the Chl b absorption (19,21) and decreases more slowly than the area from 663 to 750 nm, which corresponds to the Chl a absorption (Fig. 8B).…”