In vitro reconstitution of the photosystem I light-harvesting complex LHCI-730: Heterodimerization is required for antenna pigment organization

Abstract

Here we describe the in vitro reconstitution of photosystem I light-harvesting complexes with pigments and proteins (Lhca1 and Lhca4) obtained by overexpression of tomato Lhca genes in Escherichia coli. Using Lhca1 and Lhca4 individually for reconstitution results in monomeric pigmentproteins, whereas a combination thereof yields a dimeric complex. Interactions of the apoproteins is highly specific, as reconstitution of either of the two constituent proteins in combination with a light-harvesting protein of photosystem II does not result in dimerization. The reconstituted Lhca1͞4, but not complexes obtained with either Lhca1 or Lhca4 alone, closely resembles the native LHCI-730 dimer from tomato leaves with regard to spectroscopic properties, pigment composition, and stoichiometry. Monomeric complexes of Lhca1 or Lhca4 possess lower pigment͞protein ratios, indicating that interactions of the two subunits not only facilitates pigment reorganization but also recruitment of additional pigments. In addition to higher averages of chlorophyll a͞b ratios in monomeric complexes than in LHCI-730, comparative f luorescence and CD spectra demonstrate that heterodimerization involves preferential ligation of more chlorophyll b.Precise assembly and alignment of pigments with the various proteins encoded by a family of nuclear Lhc genes underly the formation of the light-harvesting complexes (LHCs) of thylakoid membranes, enabling the collection of solar energy and its transmission to the two photochemically active reaction centers. Although the major LHCII has been analyzed in detail with respect to protein and pigment composition and organization (1), information about LHCs of photosystem I (PSI) are limited, mostly because they are difficult to isolate abundantly in an intact state. The original finding that four proteins of about 21 to 24 kDa form the LHCI (2) is now widely accepted, and the respective genes have been identified, cloned, and sequenced (3, 4). Recently, closely related photosystem I antenna components have been identified in red algae (5, 6). In vascular plants, there are two major LHCI subfractions with different protein compositions and fluorescence properties (7-9). One, LHCI-680, is enriched in polypeptides of 24 and 23 kDa (Lhca3 and Lhca2, respectively), has characteristic 77-K fluorescence at 680 nm and a low density in sucrose gradients, and is regarded as monomeric also on the basis of electrophoretic mobility. The LHCI-680 complex can be resolved into two green bands, one enriched in Lhca2 and the other in Lhca3, showing that both proteins are pigment binding (8, 10). The second complex, LHCI-730, exhibits 77-K fluorescence around 730 nm, has a higher sedimentation coefficient, is associated with proteins of 22 and 21 kDa (Lhca1 and Lhca4, respectively), and is considered to be dimeric (7-11). Further dissection of the LHCI-730 complex has not been achieved, leaving open the question of the extent to which both constitutent apoproteins function in pigment binding and whether the complex i...

The Nature of a Chlorophyll Ligand in Lhca Proteins Determines the Far Red Fluorescence Emission Typical of Photosystem I

“…The close relation between Lhca2 and Lhca4 is confirmed by sequence analysis, which shows 55% identity and 75% homology (14). Surprisingly, the distribution of red forms does not fit into the picture, because these are associated mainly with Lhca3 and Lhca4 (fluorescence emission at 725-730 nm), whereas Lhca1 and Lhca2 have emission at higher energy (702 nm) (12,13,15). Based on high homology between proteins with different content in red forms, a sequence motif associated with the presence of lowest absorption band, common to Lhca3 and Lhca4 and not present in Lhca1 and Lhca2, should possibly be detected.…”

Conformational switching explains the intrinsic multifunctionality of plant light-harvesting complexes

“…The low-energy emission of the Lhca1/4 dimer is known to originate from the red Chls of the Lhca4 monomeric subunit (30). The role of Lhca1 in the observed spectral dynamics of Lhca1/4, and whether similar dynamics are exhibited when Lhca1 is absent, was of interest.…”

“…The degree of mixing and accordingly the emission maximum strongly depend on the local protein environment and in particular on the nature of the ligand of Chl 603 (22). Lhca1 and Lhca2, which contain the ligand histidine (H), have low-temperature emission maxima at 690 and 701 nm, respectively, whereas the ligand asparagine (N) in Lhca3 and Lhca4 assists in shifting the maxima to 725 and 732 nm, respectively (29,30). Changing in Lhca4 the ligand of Chl 603 from an N to an H, thus producing the mutant Lhca4-NH, shifts the low-temperature emission maximum to 686 nm, while an additional emission band is present at approximately 702 nm (22).…”

Structural Modeling of the Lhca4 Subunit of LHCI-730 Peripheral Antenna in Photosystem I Based on Similarity with LHCII

“…Lhca1 seems to possess the less red-shifted spectral forms (684 nm) (14,15), which is in agreement with its close relatedness to a minor lightharvesting polypeptide of PSII, CP29. On the contrary, the Lhca4 binds the "reddest" pigments (9,16).…”