1902
J. Phys. Chem. B 1997, 101, 1902-1909
Ultrafast Spectroscopy of Trimeric Light-Harvesting Complex II from Higher Plants J. P. Connelly,† M. G. Mu1 ller,† M. Hucke,† G. Gatzen,† C. W. Mullineaux,‡,§ A. V. Ruban,‡ P. Horton,‡ and A. R. Holzwarth*,† Max-Planck-Institut fu¨ r Strahlenchemie, D-45470 Mu¨ lheim an der Ruhr, Germany, and Robert-Hill-Institute, Department of Molecular Biology and Biotechnology, UniVersity of Sheffield, Sheffield S10 2TN, U.K. ReceiVed: July 2, 1996; In Final Form: NoVember 20, 1996X
Time-resolved femtosecond transient absorption measurements have been carried out at room temperature on light-harvesting chlorophyll a/b protein complex of photosystem II (LHC II) trimers prepared from spinach. Exciting in the chlorophyll (Chl) b region at 650 nm with very low intensity, virtually annihilation-free twocolor transient absorption measurement of the kinetics over 100 ps, between 645 and 690 nm, yield global lifetimes of 175 fs, 625 fs, and 5 ps and a long component (g790 ps) where the three fastest lifetimes reflect Chl b to Chl a energy transfer. Using a camera detection system, kinetics over 400 ps at still low annihilation levels and with much higher spectral resolution have been obtained. Short lifetime components of 180 fs, 480 fs, and 6 ps are comparable with the two-color data, but in addition, 34 and 85 ps components with small amplitudes are resolved and a long component (3.6 ns) is fixed at the longest lifetime value determined by fluorescence. Annihilation statistics have been calculated to compare these and earlier results. On the basis of these results and recent electron diffraction structural data, a preliminary three-pool Chl a, three-pool Chl b kinetic model is proposed. The possible influence of variable xanthophyll composition on quenching in LHC II preparations isolated from light- and dark-adapted leaves has been investigated using time-resolved picosecond fluorescence at room temperature. Global lifetimes of 5 ps, 170 ps and 3.6 ns, the lifetimes of the terminal LHC II excited state, were obtained. No discernable quenching effect due to the presence of zeaxanthin was observed.
Introduction The first few tens of picoseconds of plant photosynthesis are dependent on the efficiency and sophistication of light-harvesting complexes that absorb light and regulate the flow of energy to the reaction centers.1 These antenna complexes also serve, on the one hand, to broaden the absorption spectrum for solar radiation used by photosynthesis and, on the other hand, provide photoprotection2 and quenching mechanisms,3 the precise workings of which are only partially elucidated, that compensate for environmental variations. In higher plants and green algae, photosystem II is serviced by a group of chlorophyll (Chl) a/b antenna complexes4 of which the outermost antenna, (lightharvesting Chl a/b protein complex of photosystem II, LHC II) is the most abundant and has received the most attention. LHC II is a member of the family of chl a/b binding proteins that show high-sequence homology in a number of highly conserved sequences associated with protein-pigment interactions.5 The binding proteins’ structures and spectral properties appear to be sufficiently similar to provide a natural set of complexes to compare against the major LHC II complex and in the case of LHC II c (CP 26) (nomenclature is discussed by Jansson4); where several analogous Chls are absent, conclusions about the identity and arrangement of some pigments have been deduced by comparison of their spectra.6 In addition to their function in light absorption, the pigments, although not covalently bound to the protein, play a vital role in defining and maintaining the LHC II structure5,7 as well as imparting a degree of flexibility * Author to whom all correspondence should be addressed. E-mail:
[email protected]. FAX: (+49) 208 306 3951. † Max-Planck-Institut fu ¨ r Strahlenchemie. ‡ Robert-Hill-Institute. § Present address: Department of Biology, University College, London WC1E 6BT, U.K. X Abstract published in AdVance ACS Abstracts, February 15, 1997.
S1089-5647(96)01965-7 CCC: $14.00
to the system that may be significant in nonphotochemical quenching.8 As a result, LHC II does not yield up its complexity easily and a simultaneous understanding of its structure and function is required. LHC II shows a 3-fold symmetry motif in 2D crystals and it is also likely that the trimer is the basic LHC II unit in ViVo. The structures of the trimer and monomer subunits of LHC II have been resolved to 3.4 Å by Ku¨hlbrandt and co-workers by electron diffraction.9 Of the monomer, the major part of the 26 kDa protein structure and the positions of 12 Chl (probably 7 Chl a and 5 Chl b) and two luteins are well determined. Tantalizingly, what remains unresolved, the precise identity and orientation of the Chls and the positions of the most labile pigments (at least one xanthophyll and possibly a one or two more Chls per monomer) and terminal sections of the protein, is of great significance to the function of LHC II. The electron crystallography data indicate that the Chls are roughly located in two layers, parallel to the membrane plane and oriented with their porphyrin ring planes roughly perpendicular to the plane of the membrane. The shortest interpigment distances range from 4 to 5 Å between tetrapyrrole rings of the nearest neighbor Chls and between the luteins and closest Chls. On the basis of this proximity and the observed rapid transfer of spectral excitation from Chl b to Chl a, hypothetical assignments of the closest pairs of Chls to be Chl a-Chl b-coupling complexes and the Chls closest to the luteins to be Chl a molecules,9 thus enabling a Dexter electron exchange mechanism to prevent triplet Chl formation, seems reasonable at first glance but may not give the entirely correct assignment. The functional aspects of LHC II, in particular the energy transfer kinetics and quenching mechanisms, have been studied using steady state and time-resolved spectroscopy on a number of different preparations, including monomers, trimers, and higher aggregates. Steady state spectroscopy especially has been © 1997 American Chemical Society
Trimeric Light-Harvesting Complex II from Plants
J. Phys. Chem. B, Vol. 101, No. 10, 1997 1903
TABLE 1: Summary of Some Published LHC II Ultrafast Kinetic Data, Experimental Conditions and Calculated Annihilation Levels
ref
intensity/ hn/cm2/pulse
Bittner et al. 199519 Bittner et al. 199418
(6 × 1013)(4 × 1014) at 640 nm (8 × 1013)(5 × 1014) at 645 nm
beam diameter/ mm 0.13 0.13
Pa˚lsson et al. at 650, 665 nm 199415 Du et al. 199416 Mullineaux et al. 199321 Kwa et al. 199220
1.4 × 1013 at 640, 650 nm 6 × 1010 at 663 3 × 1013 at 650, 665, 680 nm
Eads et al. 198917 Ide et al. 198722
0.5 × 1014 5 × 1014 at 650 nm 109 at 605 nm
OD/mm 0.3 at 650 nm 0.6 at 645 nm
path photons length/ annhilationa/ % abs/trimer/pulse mm 2 10
14 57 19 63
0.3 2.1 0.4 2.7
0.55 at 650 nm 1 1 0.13
1
0.5 at 650 nm 0.1 at 680 nm 0.4 at 674 nm 0.5 at 650 nm
1
3
0.07
35 ps 13, 260 ps, 2.1, 4.3 ns 2-6, 14-36 ps, >100 ps
