Jante M. Salverda

Energy transfer in the peridinin chlorophyll-a protein of Amphidinium carterae studied by polarized transient absorption and target analysis.

The peridinin chlorophyll-a protein (PCP) of dinoflagellates differs from the well-studied light-harvesting complexes of purple bacteria and green plants in its large (4:1) carotenoid to chlorophyll ratio and the unusual properties of its primary pigment, the carotenoid peridinin. We utilized ultrafast polarized transient absorption spectroscopy to examine the flow of energy in PCP after initial excitation into the strongly allowed peridinin S2 state. Global and target analysis of the isotropic and anisotropic decays reveals that significant excitation (25-50%) is transferred to chlorophyll-a directly from the peridinin S2 state. Because of overlapping positive and negative features, this pathway was unseen in earlier single-wavelength experiments. In addition, the anisotropy remains constant and high in the peridinin population, indicating that energy transfer from peridinin to peridinin represents a minor or negligible pathway. The carotenoids are also coupled directly to chlorophyll-a via a low-lying singlet state S1 or the recently identified SCT. We model this energy transfer time scale as 2.3 +/- 0.2 ps, driven by a coupling of approximately 47 cm(-1). This coupling strength allows us to estimate that the peridinin S1/SCT donor state transition moment is approximately 3 D.

Energy Transfer in Light-Harvesting Complexes LHCII and CP29 of Spinach Studied with Three Pulse Echo Peak Shift and Transient Grating

Three pulse echo peak shift and transient grating (TG) measurements on the plant light-harvesting complexes LHCII and CP29 are reported. The LHCII complex is by far the most abundant light-harvesting complex in higher plants and fulfills several important physiological functions such as light-harvesting and photoprotection. Our study is focused on the light-harvesting function of LHCII and the very similar CP29 complex and reveals hitherto unresolved excitation energy transfer processes. All measurements were performed at room temperature using detergent isolated complexes from spinach leaves. Both complexes were excited in their Chl b band at 650 nm and in the blue shoulder of the Chl a band at 670 nm. Exponential fits to the TG and three pulse echo peak shift decay curves were used to estimate the timescales of the observed energy transfer processes. At 650 nm, the TG decay can be described with time constants of 130 fs and 2.2 ps for CP29, and 300 fs and 2.8 ps for LHCII. At 670 nm, the TG shows decay components of 230 fs and 6 ps for LHCII, and 300 fs and 5 ps for CP29. These time constants correspond to well-known energy transfer processes, from Chl b to Chl a for the 650 nm TG and from blue (670 nm) Chl a to red (680 nm) Chl a for the 670 nm TG. The peak shift decay times are entirely different. At 650 nm we find times of 150 fs and 0.5-1 ps for LHCII, and 360 fs and 3 ps for CP29, which we can associate mainly with Chl b <--> Chl b energy transfer. At 670 nm we find times of 140 fs and 3 ps for LHCII, and 3 ps for CP29, which we can associate with fast (only in LHCII) and slow transfer between relatively blue Chls a or Chl a states. From the occurrence of both fast Chl b <--> Chl b and fast Chl b --> Chl a transfer in CP29, we conclude that at least two mixed binding sites are present in this complex. A detailed comparison of our observed rates with exciton calculations on both CP29 and LHCII provides us with more insight in the location of these mixed sites. Most importantly, for CP29, we find that a Chl b pair must be present in some, but not all, complexes, on sites A(3) and B(3). For LHCII, the observed rates can best be understood if the same pair, A(3) and B(3), is involved in both fast Chl b <--> Chl b and fast Chl a <--> Chl a transfer. Hence, it is likely that mixed sites also occur in the native LHCII complex. Such flexibility in chlorophyll binding would agree with the general flexibility in aggregation form and xanthophyll binding of the LHCII complex and could be of use for optimizing the role of LHCII under specific circumstances, for example under high-light conditions. Our study is the first to provide spectroscopic evidence for mixed binding sites, as well as the first to show their existence in native complexes.

Low-Intensity Pump-Probe Measurements on the B800 Band of Rhodospirillum Molischianum

We have measured low-intensity, polarized one-color pump-probe traces in the B800 band of the light-harvesting complex LH2 of Rhodospirillum molischianum at 77 K. The excitation/detection wavelength was tuned through the B800 band. A single-wavelength and a global target analysis of the data were performed with a model that accounts for excitation energy transfer among the B800 molecules and from B800 to B850. By including the anisotropy of the signals into the fitting procedure, both transfer processes could be separated. It was estimated in the global target analysis that the intra-B800 energy transfer, i.e., the hopping of the excitation from one B800 to another B800 molecule, takes approximately 0.5 ps at 77 K. This transfer time increases with the excitation/detection wavelength from 0.3 ps on the blue side of the B800 band to approximately 0.8 ps on the red side. The residual B800 anisotropy shows a wavelength dependence as expected for energy transfer within an inhomogeneously broadened cluster of weakly coupled pigments. In the global target analysis, the transfer time from B800 to B850 was determined to be approximately 1.7 ps at 77 K. In the single-wavelength analysis, a speeding-up of the B800 --> B850 energy transfer rate toward the blue edge of the B800 band was found. This nicely correlates with the proposed position of the suggested high-exciton component of the B850 band acting as an additional decay channel for B800 excitations.

Energy Transfer in the B800 Ring of Light Harvesting Antenna Lh2 of Purple Bacteria rps. Acidophila and rs. Molischianum Probed by Three Pulse Echo Peakshift

The light-harvesting antenna protein LH2 of photosynthetic purple bacteria has been the subject of intensive study since the structures of the LH2 complexes of Rhodospirillum molischianum [1] and Rhodopseudomonas acidophila [2] were resolved a few years ago. The LH2 complex is a ring shaped structure with 8 [1] or 9 [2] subunits, each of which binds one bacteriochlorophyll pigment absorbing at 800 nm and two absorbing at 850 nm.

Polarized and Colored Photon Echo Measurements on the LH2 Antenna of RPS. Acidophila and RS. Molischianum

The reaction center (RC) of purple bacteria is surrounded by two types of light harvesting antennae, the core antenna LH1 enclosing the RC and the peripheral antenna LH2. Both LH1 and LH2 consist of a ring of polypeptides binding bacteriochlorophyll (Bchl) and carotenoid (Car) pigments. The structure of LH2 was resolved to 2.5 A a few years ago, for both Rhodopseudomonas acidophila [1] and Rhodospirillum molischianum [2]. The ring was found to contain 9 or 8 subunits respectively, each of which consists of an α and β polypeptide, binding 3 Bchls and at least 1 Car. Of the Bchls, one absorbs at 800 nm, and the other two, which are very close to each other and their next neighbors (9 A center to center distance), at 850 nm. The 9/8 B800 Bchls are separated from each other by 21 A, and from the B850 BchIs in their own subunit by only slightly less.