Excitonic Energy Level Structure and Pigment−Protein Interactions in the Recombinant Water-Soluble Chlorophyll Protein. II. Spectral Hole-Burning Experiments

Abstract

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).

Probing environment fluctuations by two-dimensional electronic spectroscopy of molecular systems at temperatures below 5 K

“…Application of such approaches to specific photosynthetic aggregates revealed the spectral density for a range of significant photosynthetic systems. [32][33][34][35][36][37][38] We note that HB spectroscopy (via zero-phonon action spectra, i.e., burning of resonant holes at the lowest energy state using constant fluence) can also provide information about static (inhomogeneous) broadening, while the width of resonant holes (burned at various spectral regions) can reveal information on excitation energy transfer (EET) and chargetransfer processes in model and complex biological systems. It should be noted that the few K temperature also allows following of the photosynthesis-relevant processes, since basic light harvesting, excitation transport and charge separation still take place in these conditions.…”

“…The parameters of our model are inspired by the HB and FLN spectroscopies of a recombinant water-soluble chlorophyll protein (WSCP). 36,37,41 It has been shown recently that both WSCP from Lepidium virginicum (LvWSCP) and cauliflower (Brassica oleracea L. var. botrys -CaWSCP) have four chlorophylls in a tetramer, i.e., two weakly coupled dimers 42 in which uncorrelated EET was observed.…”

Protein and solvent dynamics of the water-soluble chlorophyll-binding protein (WSCP)

“…A recombinant type of WSCP from cauliflower was found to contain 2 chlorophylls (class-IIa), while WSCP from Lepidium virginicum binds 4 chlorophylls per tetramer (class-IIb). Recombinant class-IIa WSCP binding only two chlorophylls has been used to investigate pigmentprotein interactions and excitation energy transfer by a number of spectroscopic techniques including timeresolved absorption and fluorescence experiments, spectral line-narrowing and 2D electronic spectroscopy [10][11][12][13][14].…”

Identification of genes encoding photoconvertible (Class I) water-soluble chlorophyll-binding proteins from Chenopodium ficifolium

“…[18][19][20][21][22][23] Furthermore, various molecular genetic analyses on recombinant Class II WSCPs have been performed. 18,[20][21][22][23][24][25][26][27][28][29][30][31][32] On the other hand, CaWSCP is the only gene encoding a Class I WSCP identified to date and few studies on the molecular genetics of the protein have been performed thus far. 33,34) Identification of novel genes encoding Class I WSCPs is essential for understanding the molecular diversity of the protein.…”