A gene for a light-harvesting chlorophyll (Chl) a/b-binding protein (LHCP) from pea (Pisum sativum L.) has been cloned in a bacterial expression vector. Bacteria (Escherichia coli) transformed with this construct produced up to 20% of their protein as pLHCP, a derivative of the authentic precursor protein coded for by the pea gene with three amino-terminal amino acids added and-or exchanged, or as a truncated LHCP carrying a short amino-terminal deletion into the mature protein sequence. Following the procedure of Plumley and Schmidt (1987, Proc. Natl. Acad. Sci. USA84, 146-150), all bacteria-produced LHCP derivatives can be reconstituted with acetone extracts from pea thylakoids or with isolated pigments to yield pigment-protein complexes that are stable during partially denaturing polyacrylamide-gel electrophoresis. The spectroscopic properties of these complexes closely resemble those of the light-harvesting complex associated with photosystem II (LHCII) isolated from pea thylakoids. The pigment requirement for the reconstitution is highly specific for the pigments found in native LHCII: Chl a and b as well as at least two out of three xanthophylls are necessary. Varying the Chl a:Chl b ratios in the reconstitution mixtures changes the yields of complex formed but not the Chl a:Chl b ratio in the complex. We conclude that LHCP-pigment assembly in vitro is highly specific and that the complexes formed are structurally similar to LHCII. The N-terminal region of the protein can be varied without affecting complex formation and therefore does not seem to be involved in pigment binding.

The reliability of procedures for extracting the distance distribution between spins from the dipolar evolution funetion is studied with particular emphasis on broad distributions. A new numerically stable procedure for fitting distance distributions with polynomial interpolation between sampling points is introduced and compared to Tikhonov regulafization in the dipolar frequency and distante domains and to approximate Pake transformation. Distante distributions with only narrow peaks are most reliably extracted by distance-domain Til~aonov regularization, while frequency-domain Tikhonov regularization is favorable for distributions with only broad peaks. For the quantification of distributions by their mean distante and variante, Hermite polynomial interpolation provides the best results. Distributions that contain both broad and narrow peaks are most difficult to analyze. In this case a high signal-to-noise ratio is strietly required and approximate Pake transformation should be applied. A proeedure is given for renormalizing primary expe¡ data from protein preparations with slightly different degrees of spin labeUing, so that they can be compared directly. Performance of all the data analysis procedures is demonstrated on experimental data for a shape-persistent biradical with a label-to-label distante of 5 nm, for a [2]catenane with a broad distante distribution, and fora doubly spin-labelled double mutant of plant light harvesting complex II.

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.

A gene coding for water-soluble chlorophyll-binding protein (WSCP) from Brassica oleracea var. Botrys has been used to express the protein, extended by a hexahistidyl tag, in Escherichia coli. The protein has been refolded in vitro to study its pigment binding behavior. Recombinant WSCP was found to bind two chlorophylls (Chls) per tetrameric protein complex but no carotenoids in accordance with previous observations with the native protein [Satoh, H., Nakayama, K., Okada, M. (1998) J. Biol. Chem. 273, 30568-30575]. WSCP binds Chl a, Chl b, bacteriochlorophyll a, and the Zn derivative of Chl a but not pheophytin a, indicating that the central metal ion in Chl is essential for binding. WSCP also binds chlorophyllides a and b and even the more distant Chl precursor Mg-protoporphyrin IX; however, these pigments fail to induce oligomerization of the protein. We conclude that the phytol group in bound Chl plays a role in the formation of tetrameric WSCP complexes. If WSCP in fact binds Chl or its derivative(s) in vivo, the lack of carotenoids in pigmented WSCP raises the question of how photooxidation, mediated by triplet-excited Chl and singlet oxygen, is prohibited. We show by spin-trap electron-paramagnetic resonance that the light-induced singlet-oxygen formation of WSCP-bound Chl is lower by a factor of about 4 than that of unbound Chl. This as-yet-unknown mechanism of WSCP to protect its bound Chl against photooxidation supports the notion that WSCP may function as a transient carrier of Chl or its derivatives.