No abstract

Photosystem I with its full antenna complement (PSI-LHCI) has been prepared by mild detergent solubilization with octyl beta-D-glucopyranoside from maize thylakoids. A preliminary polypeptide analysis is presented. At room temperature, the steady-state fluorescence derives from an almost perfectly thermalized state, as demonstrated by a Stepanov analysis, in which about 90% of the excited states are associated with the red chlorophyll spectral forms absorbing above 700 nm. Equilibration is temperature-sensitive and is lost at T < 200 K. A careful analysis of fluorescence between 75 and 280 K clearly demonstrates the presence of at least three red chlorophyll spectral forms with emission maxima at 720, 730, and 742 nm, the absorption origin bands of which have been calculated at 714, 725, and 738 nm. On the basis of a minor deviation from thermal equilibration around 695 nm, it is suggested that at least 3-4 antenna chlorophylls, with an average absorption near 695 nm, are strongly coupled to P700. Thermodynamic analysis of absorption and fluorescence spectra indicates that the equilibrium, absorption-weighted excited state population of the P700 dimer is around 0.013 assuming that the low-energy exciton state possesses all the oscillator strength. The average free energy for excitation transfer from antenna to P700 is thus calculated to be -0.26 kT at room temperature. This indicates that P700 is almost isoenergetic with its antenna at room temperature when the red forms are taken fully into account. From the calculated excited state population of P700, we estimate that the primary charge separation rate in PSI is 1-2 ps-1.

The intact photosystem I of maize containing its full antenna complement (PSI-200) has been purified and fractionated into the core and outer antenna (LHCI) components. It is demonstrated by absorption and fluorescence spectroscopy that at least 80% of the long wavelength absorbing antenna pigments (red forms) are located in LHCI. Absorption spectra in the Qy region of all three preparations were measured between 72 and 300 K and subjected to a thermal broadening analysis. Data are interpreted in the linear electron-phonon coupling assumption, and the average optical reorganization energy (Snum) for the bulk pigment band and the red absorption tail determined. A marked asymmetry in Snum values across the absorption band is demonstrated. The bulk pigments in all three preparations have rather low values, in the range of 15-25 cm-1, suggesting that Stokes shifts for the absorption forms are in the 1. 5-3 nm range. On the other hand the red forms have markedly greater reorganization energies. While a direct thermal analysis of the red tail indicates minimum Snum values of around 60 cm-1, when the contribution of the red tail of the bulk pigments is corrected for in LHCI, the more reliable value of 110 cm-1 is obtained. These high Snum values for the red pigment forms suggest that they have unusually wide homogeneously broadened absorption bands and large Stokes shifts (6-11 nm).

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.

The chlorophyll-protein complexes that form the antenna system of photosystem II have been purified and analyzed in terms of the commonly observed chlorophyll spectral forms. With the exception of chlorophyll b, which is known to be associated with the complexes comprising the outer antenna (LHCII, CP24, CP26, CP29), the spectral forms occur with similar absorption maxima and are present in rather similar amounts in each of the antenna complexes. On the basis of the published chlorophyll stoichiometries for the complexes in photosystem II antenna, the distribution of the spectral forms in a "reconstituted" antenna has been determined. These data were used to calculate the equilibrium population of excited states within the various chlorophyll-protein complexes within photosystem II. This was compared with the light absorption capacity of each of the complexes in the "reconstituted" antenna. The ratio of these two parameters (excited-state equilibrium distribution/absorption capacity) was determined to be 1.21 for the inner (core) antenna and 0.88 for LHCII. The standard free energy change for exciton transfer from the outer to the inner antenna was calculated to be -0.17 kcal mol-1. It is concluded that the photosystem II antenna is arranged as a very shallow funnel.