Time-local and time-nonlocal theories are used in combination with optical spectroscopy to characterize the water-soluble chlorophyll binding protein complex (WSCP) from cauliflower. The recombinant cauliflower WSCP complexes reconstituted with either chlorophyll b (Chl b) or Chl a/Chl b mixtures are characterized by absorption spectroscopy at 77 and 298 K and circular dichroism at 298 K. On the basis of the analysis of these spectra and spectra reported for recombinant WSCP reconstituted with Chl a only (Hughes, J. L.; Razeghifard, R.; Logue, M.; Oakley, A.; Wydrzynski, T.; Krausz, E. J. Am. Chem. Soc. U.S.A. 2006, 128, 3649), the "open-sandwich" model proposed for the structure of the pigment dimer is refined. Our calculations show that, for a reasonable description of the data, a reduction of the angle between pigment planes from 60 degrees of the original model to about 30 degrees is required when exciton relaxation-induced lifetime broadening is included in the analysis of optical spectra. The temperature dependence of the absorption spectrum is found to provide a unique test for the two non-Markovian theories of optical spectra. Based on our data and the 1.7 K spectra of Hughes et al. (2006), the time-local partial ordering prescription theory is shown to describe the experimental results over the whole temperature range between 1.7 K and room temperature, whereas the alternative time-nonlocal chronological ordering prescription theory fails at high temperatures. Modified-Redfield theory predicts sub-100 fs exciton relaxation times for the homodimers and a 450 fs time constant in the heterodimers. Whereas the simpler Redfield theory gives a similar time constant for the homodimers, the one for the heterodimers deviates strongly in the two theories. The difference is explained by multivibrational quanta transitions in the protein which are neglected in Redfield theory.

Persistent spectral hole burning at 4.5 K has been used to investigate the excitonic energy level structure and the excited state dynamics of the recombinant class-IIa water-soluble chlorophyll-binding protein (WSCP) from cauliflower. The hole-burned spectra are composed of four main features: (i) a narrow zero-phonon hole (ZPH) at the burn wavelength, (ii) a number of vibrational ZPHs, (iii) a broad low-energy hole at ~665 and ~683 nm for chlorophyll b- and chlorophyll a-WSCP, respectively, and (iv) a second satellite hole at ~658 and ~673 nm for chlorophyll b- and chlorophyll a-WSCP, respectively. The doublet of broad satellite holes is assigned to an excitonically coupled chlorophyll dimer. The lower-energy holes at ~665 and ~683 nm for chlorophyll b- and chlorophyll a-WSCP, respectively, represent the lower exciton states. Taking into account the parameters of electron-phonon coupling, the lower exciton state can be assigned as the fluorescence origin. The lower exciton state is populated by two processes: (i) exciton relaxation from the higher exciton state and (ii) vibrational relaxation within the lower exciton state. Assuming identical site energies for the two excitonically coupled chlorophyll molecules, the dipole-dipole interaction energy J is directly determined to be 85 and 100 cm(-1) for chlorophyll b- and chlorophyll a-WSCP, respectively, based on the positions of the satellite holes. The Gaussian low-energy absorption band identified by constant fluence hole burning at 4.5 K has a width of ~150 cm(-1) and peaks at 664.9 and 682.7 nm for chlorophyll b- and chlorophyll a-WSCP, respectively. The action spectrum is broader and blue-shifted compared to the fluorescent lower exciton state. This finding can be explained by a slow protein relaxation between energetically inequivalent conformational substates within the lowest exciton state in agreement with the results of Schmitt et al. (J. Phys. Chem. B2008, 112, 13951).

The crystal structure of the class IIb water-soluble chlorophyll binding protein (WSCP) from Lepidium virginicum is used to model linear absorption and circular dichroism spectra as well as excited state decay times of class IIa WSCP from cauliflower reconstituted with chlorophyll (Chl) a and Chl b. The close agreement between theory and experiment suggests that both types of WSCP share a common Chl binding motif, where the opening angle between pigment planes in class IIa WSCP should not differ by more than 10 degrees from that in class IIb. The experimentally observed (Schmitt et al. J. Phys. Chem. B 2008, 112, 13951) decrease in excited state lifetime of Chl a homodimers with increasing temperature is fully explained by thermally activated superradiance via the upper exciton state of the dimer. Whereas a temperature-independent intersystem crossing (ISC) rate is inferred for WSCP containing Chl a homodimers, that of WSCP with Chl b homodimers is found to increase above 100 K. Our quantum chemical/electrostatic calculations suggest that a thermally activated ISC via an excited triplet state T4 is responsible for the latter temperature dependence.

Based on these results it is inferred that photosynthetic dioxygen formation comprises the cleavage of at least one hydrogen bond.

The present study describes the fluorescence emission properties of recombinant water-soluble chlorophyll (Chl) protein (WSCP) complexes reconstituted with either Chl a or Chl b alone (Chl a only or Chl b only WSCP, respectively) or mixtures of both pigments at different stoichiometrical ratios. Detailed investigations were performed with time and space correlated ps fluorescence spectroscopy within the temperature range from 10 to 295 K. The following points were found: (a) The emission spectra at room temperature (295 K) are well characterized by bands with a dominating Lorentzian profile broadened due to phonon scattering and peak positions located at 677, 684 and 693 nm in the case of Chl a only WSCP and at 665, 675 and 689 nm for Chl b only WSCP. In addition, all spectra contain minor bands in the longer wavelength region. (b) The emission spectra at 10 K of samples suspended in buffer containing 50% glycerol are dominated by bands peaking at 668 nm for Chl b only WSCP and at 685 nm for Chl a only WSCP and samples reconstituted with mixtures of Chl a and Chl b. (c) At 10 K and in buffer with 50% glycerol the decay kinetics of WSCP samples with Chl a only are dominated by a component with a time constant of 6.2 (+/-0.2) ns at 685 nm while those of WSCP containing mixtures of Chl a and Chl b are characterized by a slightly shorter value of 6.0 (+/-0.2) ns. WSCP containing Chl b only exhibits a distinctly longer value of 7.0 (+/-0.3) ns at an emission wavelength of 668 nm. (d) The decay associated emission spectra at 10 K of all samples exhibit at least 3 decay components with time constants of 80-120 ps, 2-4 ns and 6-7 ns in 50% glycerol. These results are consistently described within the framework of our previously presented model (J. Phys. Chem. B 2007, 111, No. 46, 13325; J. Phys. Chem. B 2007, 111, No. 35, 10487) , for the structural motifs of chlorophyll binding to the tetrameric protein matrix of WSCP. It is shown that formation of strongly coupled open sandwich dimers does not lead to quenching of 1Chl a* or 1Chl b*.