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...

AB96, a gene encoding a Pisum sativum chlorophyll a/b binding protein [Coruzzi et al. (1983) J. Biol. Chem. 258, 1399-1402], can be expressed in Escherichia coli and reconstituted with pigments by the procedure described by Plumley and Schmidt [(1987) Proc. Natl. Acad. Sci. U.S.A. 84, 146-150]. Following purification by polyacrylamide gel electrophoresis, the reconstituted pigment-protein complex (CP2) is shown to have similar pigment-binding characteristics to native CP2 complexes isolated from thylakoid membranes. Therefore, the AB96 gene product contains binding sites for chlorophylls a and b and xanthophylls, all of which are necessary for optimal reconstitution in vitro. Absorption, fluorescence, and circular dichroism spectroscopy indicate that the pigments are oriented accurately and that chlorophylls a and b are adjoined for energy transfer. Studies with proteins produced after deletion mutagenesis of AB96 indicate that NH2-terminal amino acids 1-21 and COOH-terminal amino acids 219-228 do not play a role in pigment binding. In contrast, amino acids 50-57 and 204-212 (encompassing one of three conserved histidine residues) are essential for reconstitution. Residues near the presumed NH2- and COOH-terminal alpha-helix boundaries (22-49 and 213-218, respectively) affect the stability of reconstituted CP2 during electrophoresis at 4 degrees C. Correlation of diminished chlorophyll a binding with disappearance of a negative circular dichroism near 684 nm suggests that amino acids 213-218 near the COOH-terminal boundary of the third membrane-spanning helix affect the binding of some chlorophyll a molecules.

Analyses were made of the effects of extraction of the 17,24 kilodalton extrinsic proteins from spinach versus wheat photosystem II (PSII) membranes on Ca abundance and 02 evolution capacity determined in the absence and presence of either ClO or Ca22. Extraction of these proteins from spinach PSII routinely diminished steady state 02 evolution by about 70% when assayed in the presence ofsufficient Cl1. Additionally, 02 evolution of 17,24 kilodalton-less spinach PSI1 membranes showed about 2-fold more enhancement by Ca22 than by Cl-during assay. When the same extraction and assay procedures were applied to wheat PSII membranes, we observed, in contrast to 17,24 kilodalton-less spinach PSII, only about 50% inhibition of 02 evolution and about 2-fold greater enhancement by Cl-than by Ca21. Irrespective of differences in the magnitude of enhancement of 02 evolution by Ca2' versuC a-in spinach versus wheat, the K., values for Cl-(about 1.7 millimolar) and Ca2l (about 1.5 millimolar) were similar for both type preparations. The abundance of Ca specifically associated with fully functional PSII (about 2 and about 3 Ca/200 chlorophyll for spinach and wheat, respectively) was diminished to about 1 per 200 chlorophyll upon 17.24 kilodalton protein depletion. Further treatment of wheat 17,24 kilodalton-less PSII in darkness with 2 molar NaClI/ millimolar ethyleneglycol-bis(O-aminoethyl ether)-N,N'-tetraacetic acid/20 micromolar A231872 made 02 evolution highly dependent on Ca2 addition, much like the 17,24 kilodaltonless spinach PSII. Analyses of this Ca2' effect on 02 evolution revealed both high (K. about 65 micromolar) and low (K. about 1.5 millimolar) affinity Ca2 sites in wheat 17,24 kilodalton-less PSII. The results suggest that during 17,24 kilodalton extraction by NaCl, spinach PSII is more susceptible than wheat PSII to loss of high affinity Ca and irreversible inhibition of 02 evolution.The realization of 02 evolving everted thylakoid vesicles (1, 21) and Triton X-100 prepared PSII membranes (3, 24), also having everted membrane orientation (14,24), has led to intensive research focused on defining the polypeptides essential for efficient functioning of the PSII/water oxidizing complex. Parallel research has been directed towards identifying which poly- 90% (4-7, 15, 16, 21, 25, 27) when determined in the presence of saturating concentrations of Cl-. Subsequently, it was shown (6,7,15,16) that addition of 10 to 20 mM Ca2+ to 17,24 kDless, Cl-sufficient spinach PSII membranes increased rates of 02 evolution as much as 4-to 6-fold to rates sometimes nearly equivalent to those observed with unextracted membranes. However, no Ca2' additions were necessary to observe substantial period-4 oscillations of the S-states with only about 25% disconnection of the S-state complexes from PSII traps (12).Conceivably, at least some of the rather large variability in extent of loss of 02 evolution following depletion of the 17,24 kD proteins could reflect inadvertent dissociation of high affinity Ca2`during extraction....

Polyphenol oxidase activity (E.C. 1.14.18.1) has been found in two enzyme species isolated from thylakoid membranes of spinach chloroplasts. The proteins were released from the membrane by sonication and purified >900-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a molecular weight of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. The higher molecular weight enzyme is the predominant form in freshly isolated preparations but on aging or further purification, the amount of lower molecular weight enzyme increases at the expense of the higher.Sonication releases polyphenol oxidase from the membrane largely in the latent state. C18 fatty acids, especially linolenic acid, are potent activators of the enzymic activity. In the absence of added fatty acids, the isolated enzyme spontaneously, but slowly, activates with time.Purified Polyphenol oxidase (o-diphenol: 02 oxidoreductase)2 has been found in chloroplasts of nearly a dozen higher plants (2,3,9,14,16,27 were reported to be interconvertible. At present, it is uncertain whether or not the thylakoid-bound and stromal forms of the enzyme are related. The enzymic activity of plant polyphenol oxidase is latent. Activation can be achieved by treating extracts or membranes with detergents (16), acid or alkali (12), denaturing agents (22), or with proteolytic enzymes such as trypsin (27) or trypsin plus carboxypeptidase a (19). In many cases, the enzyme is activated upon release from the thylakoid membrane, but there is no indication that solubilization and activation are part of the normal function of the enzyme in the chloroplast. Indeed, the only physiological activator known is the process of aging.In this paper, we report the isolation, purification, and several properties of thylakoid-bound polyphenol oxidase from spinach chloroplasts. We demonstrate that the enzyme can be liberated from the thylakoid membrane largely in the latent state, and that activity can be initiated in the bound and released forms by a group of physiologically important molecules, the fatty acids. MATERIALS AND METHODSReagents and Chemicals. Linolenic acid was purchased from Aldrich; coumaric acid from Calbiochem; lithium dodecyl sulfate from BHD Chemicals; and trypsin from Nutritional Biochemicals Corporation. The protein standards ovalbumin, chymotrypsinogen, and ribonuclease A were supplied by Pharmacia, and human IgG was supplied by Miles Laboratories. Protein standards for SDS gel electrophoresis were obtained from Bio-Rad Laboratories. The remaining reagents and buffers were purchased from Sigma.Protein Determination and Enzyme Assays. Protein was determined according to the method of Bradford (4) using bovine plasma gamma globulin as primary standard. Chl was determined in 80o acetone (2). Polyphenol oxidase activity was assayed by measuring 02 uptake coupled to the oxidation of DL-DOPA using a Clark-type electrode. Unless otherwise stated, the el...

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The structure and heterogeneity of LHC II were studied by in vitro reconstitution of apoproteins with pigments (Plumley and Schmidt 1987, Proc Natl Acad Sci 84: 146-150). Reconstituted CP 2 complexes purified by LDS-PAGE were subsequently characterized and shown to have spectroscopic properties and pigment-protein compositions and stoichiometries similar to those of authentic complexes. Heterologous reconstitutions utilizing pigments and light-harvesting proteins from spinach, pea and Chlamydomonas reinhardtii reveal no evidence of specialized binding sites for the unique C. reinhardtii xanthophyll loroxanthin: lutein and loroxanthin are interchangeable for in vitro reconstitution. Proteins modified by the presence of a transit peptide, phosphorylation, or proteolytic removal of the NH2-terminus could be reconstituted. Evidence suggests that post-translational modification are not responsible for the presence of six electrophoretic variants of C. reinhardtii CP 2. Reconstitution is blocked by iodoacetamide pre-treatment of the apoproteins suggesting a role for cysteine in pigment ligation and/or proper folding of the pigment-protein complex. Finally, no effect of divalent cations on pigment reassembly could be detected.