Reconstitution of carotenoids

G. Method for Reconstitution of Carotenoids into Reaction Centers of Rb. sphaeroides R-26.1. 240... 

Davidson Eand Cogdell RJ (1981) Reconstitution of carotenoids into the light-harvesting pigment-protein complex from the carotenoidless mutant of Rhodopseudomorms sphaeroides R-26. Biochim Biophys Acta 635 295-303... 

The reconstituted complexes were found to be very similar to their native counterparts with regard to their biochemical and spectroscopic properties. Therefore, the reconstitution of Chi a/b complexes in vitro has been exploited to yield much viuable information on the role of carotenoids in the assembly of these complexes. 

The first publication on the reconstitution of the major LHCII complex already contained some information on the role of specific carotenoids (Plumley and Schmidt, 1987). When carotenoids were altogether omitted from the reconstitution mixture, no reconstitution of stable complexes was observed, so carotenoids are essential. However, not all three carotenoids found in native LHCII were... 

Other Chi a/b complexes can be reconstituted with pigments under very similar conditions as the major LHCII complex. When reconstituting CP29 with varying combinations of carotenoids, only lutein proves to be indispensable. When either violaxanthin or neoxanthin is omitted from the pigment mixture, the complexes appear about as stable as complexes reconstituted with the full complement of carotenoids (Giuffraetal., 1996). 

Little is known about the role of carotenoids in the assembly of Chi c-containing algal light-harvesting complexes. The Chi a, c-containing LHCII of the prasinophyte Mantoniella squamata has been reconstituted in vitro from the denatured native complex. This complex contains prasinoxanthin, violaxanthin, and neoxanthin but can also bind some externally added lutein (Meyer and Wilhelm, 1993). 

Table I. Carotenoid requirement for reconstituting different Chi a/b proteins. Data from different studies are compiled on the reconstitution of Chi a/b proteins in vitro with the reduced carotenoid set shown in addition to Chi a and Chi b. Stabilities of reconstituted complexes are estimated on the basis of reconstitution yields. +-H-, complexes about as stable as the native complexes ++, complexes significantly less stable than native complexes +, complexes rather instable, must be isolated under low-stringency conditions nd, not determined lut, lutein neo, neoxanthin vio, violaxanthin.

Of course, the interaction between the membrane-spanning helices A and B that is presumably stabilized by these carotenoids may in turn influence the trimer formation of the complex. However, it is also possible that the additional carotenoid(s), known to be part of the complex from biochemical data but not visible in the crystal stmcture, stabilize trimers. This can be suggested from the observation that in the aba mutant of Arabidopsis where zeaxanthin appears to replace violaxanthin and neoxanthin, the major LHCII dissociates more easily into monomers when isolated under partially denaturing conditions (Tardy and Havaux, 1996). A closer biochemical inspection of the major LHCII in various carotenoid-deficient plant and algae mutants will be necessary to assess the impact of carotenoids on the formation of trimeric LHCII. Another experimental approach will be to study how the variation of the carotenoid components influences the reconstitution of trimeric LHCII in vitro (Hobe et al, 1994). 

Davis CM, Bustamante PLand Loach PA (1995) Reconstitution of thebacteri al core light-harvesting complexes of R/jodohader sphaeroides and Rhodospirillum rubrum with isolated alpha-and beta-polypeptides, bacteriochlorophyll a, and carotenoid. J Biol Chem 270 5793-5804... 

Jrrsakova V and Reiss-Husson F (1994) A specific carotenoid is required for reconstitution of Xh Rubrivivax gelatinosus B875 light harvesting complex from its subunit form B820. FEBS Lett 353 151-154... 

Chemical synthesis has a major part to play in the sophisticated interdisciplinary studies that are now needed to study the biological functions and actions of carotenoids, and the interactions of carotenoids with other molecules such as proteins. Essential roles in photosynthesis have been discovered for several different carotenoids, including specific geometrical isomers. Synthesis is able to provide the pure and, when appropriate, isotopically labelled carotenoids that are required for reconstitution studies, investigation of photochemistry, etc. In the field of medicine it is now clear that the provitamin A activity of p,p-carotene (3) may not be the only beneficial effect of carotenoids. Several carotenoids found in the human diet, especially lycopene (31), lutein (133) and zeaxanthin (119), could also be important in giving protection against serious disorders such as cancer, heart disease, and degenerative eye diseases. Characterization of these effects and elucidation of the mechanisms involved require substantial quantities (g to kg) of pure carotenoids these materials can only be produced by chemical synthesis. 

Phylloquinone has become a focus of a variety of structure function relationships due to its central role in electron transfer. The PhQ molecules can be extracted from PS I using dry or water-saturated diethyl ether o or using hexane containing 0.3% methanolWater-saturated ether extraction also removes a significant number of antenna chlorophylls, and aU of the carotenoids (and probably Hpids), while leaving the iron-sulfur clusters unaffected. As might be expected, both extraction procedures block room-temperature electron transfer from Aq to the iron-sulfur clusters. - Reconstitution of one PhQ fully restores electron transfer from A to the iron-sulfur clusters. Iwaki and coworkers reported that PhQ could be replaced by a variety of quinones with appropriate redox potentials. Moreover, an assortment of non-native quinones can be inserted into the PhQ binding site in In particular. 

FIGURE 3. A) Comparison of fluorescence excitation spectra for fluorescence maximum at 724 nm - 1 - PPG III in Tris-HCl buffer + 0.2% Triton X-100. 2 - sample 1 after extraction of carotenoids (heptane) and their back-reconstitution. Temperature - 100 K, B) Temperature dependence of fluorescence excitation spectra of PPG III in polyacrylamide gel for emission maximum at 721 nm. Temperatures 1 - 88 K,... 

BOUVIER F, D HARLINGUE A, HUGUENEY P, MARIN E, MARION-POLL A and CAMARA B (1996) Xanthophyll biosynthesis cloning, expression, functional reconstitution and regulation of 3-cyclohexenyl carotenoid epoxidase from pepper Capsicum annuum) , J Biol Chem, 271, 28861-7. 

The use of selective isotope replacement of carbon and hydrogen atoms in the structure of xanthophylls in combination with LHCII reconstitution should greatly aid the assignment of multiple v4 twisting bands. This assignment would help localize the areas of distortion within the carotenoid molecule and understand the possible causes of this distortion. 

Karlin-Neumann et al. [31] have presented a model for the main apoprotein of LHC II which has 3 a-helices (Chapter 11), consistent with the direct determination of the a-helical content of LHC II [32]. There is a large domain of surface-exposed protein (= 48%) this is also consistent with the electron microscopic pictures of reconstituted LHC II [18,19], While most of the Chi molecules are thought to reside in the hydrophobic membrane interior, there are insufficient histidine residues present for the co-ordination of all Chi a molecules, and the ligand for Chi b has not been recognized yet. The location of the carotenoids, so often ignored, but always a constituent of all Chl-proteins, is not established [30],... 

Sadler, G., Davis, J. and Dezman, D. 1990. Rapid extraction of lycopene and P-carotene from reconstituted tomato paste and pink grapefruit homogenates. J. Food Sci. 55 1460-1461. Sakamoto, H., Mori, H., Ojima, F., Ishiguro, Y., Arimoto, S., Imae, Y., Nanba, T., Ogawa, M. and Fukuba, H. 1994. Elevation of serum carotenoids after continual ingestion of tomato juice. J. Jpn Soc. Nutr. Food Sci. 47 93-99. 

Phylloquinone extraction and reconstitution was performed by Itoh, Iwaki and Ikegami " with digitonin PS-I particles and P700-enriched particles. The extraction process was monitored directly by the EPR spectrum ofthe photoaccumulated, A semiquinone radical with its characteristic g-value of2.004 and halfwidth of 10 G. The digitonin PS-I particle used for extraction contained 150 chlorophyll, 20 carotenoid and 2.2 phylloquinone molecules per P700. Extraction with dry ether removed -60% ofthe chloro-... 

The hexane-extracted particles that still retained one Q per P700 retained 81 % of the NADP -reduction activity ofthe unextracted control. In contrast, particles extracted with hexane-0.3% methanol had all the OQ removed and the NADP -reduction activity was completely lost. The NADP -reduction activity of the hexane/methanol-extracted particles could be reactivated by readdition of exogenous but only when the hexane extract was also added back. Exogenous OQ alone, even at a rather high concentration, could not reactivate NADP photoreduction and the hexane extract alone was also not effective, presumably because of its low Q content. The nature ofthe component in the hexane extract that contributes to reconstitution is as yet unknown. As the hexane extract contains OQ,chlorophylls,carotenoids,lipidsand other nonpolar molecules, some critical component is probably needed to ensure the correct membrane structure for binding ferredoxin and/or Fd-NADP -reductase. This conclusion is supported by the fact that activity of prior terminal acceptors such as the iron-sulfur centers do not require the hexane extract. 

---