The LH2 complexes are assembled in the cells of purple sulfur bacterium Ectothiorhodospira haloalkaliphila with inhibition of carotenoid biosynthesis

The effect of the inhibitor of carotenoid (Car) biosynthesis, diphenylamine (DPA), on the cells of the purple sulfur bacterium Ectothiorhodospira (Ect.) haloalkaliphila has been studied. There occurs an inhibition of the biosynthesis of colored Cars (≥99 %) at 71 μM DPA. Considering “empty” Car pockets (Moskalenko and Makhneva 2012) the content of Cars in the DPA-treated samples is first calculated more correctly. The total content of the colored Cars in the sample at 71 μM DPA does not exceed 1 % of the wild type. In the DPA-treated cells (membranes) a complete set of pigment-protein complexes is retained. The LH2 complex at 71 μM DPA is isolated, which is identical to the LH2 complex of the wild type in near IR absorption spectra. This suggests that the principles for assembling this LH2 complex in vivo in the absence of colored Cars remain the same. These results are in full agreement with the data obtained earlier for Allochromatium (Alc.) minutissimum (Moskalenko and Makhneva 2012). They are as follows: (1) DPA almost entirely inhibits the biosynthesis of the colored Cars in Ect. haloalkaliphila cells. (2) In the DPA-treated samples non-colored Cars are detected at 53.25 μM DPA (as traces) and at 71 μM DPA. (3) DPA may affect both phytoene synthase (at ≤71 μM DPA) and phytoene desaturase (at ≥53.25 μM DPA). (4) The assembly of LH2 complex does occur without any colored Cars.     

The role of aromatic phenylalanine residues in binding carotenoid to light-harvesting model and wild-type complexes

The mode of carotenoid (Crt) binding to polypeptide and specifying its function is as yet largely unknown. Statistical analysis of major photosystems I and II suggests that aromatic residues make up a significant part of the Crt binding pockets. Phenylalanine residues ensure approximately 25%—at some carbon atoms even up to 40%—of the total contacts with Crts. By use of an alanine-leucine model transmembrane helix that replaces the native helix of the bacterial light-harvesting complex 2 (LH2) α-subunit, we study the effects of polypeptide residues on cofactor binding in a model sequence context. Here, it is shown that phenylalanine residues located in the close vicinity of the Crts' polyene backbone significantly contribute to the binding of the Crt to the model protein. The replacement of a phenylalanine with leucine in the model helix results in significant reduction in the complexes' Crt content. This effect is strongly enhanced by the removal of a second phenylalanine in close vicinity to the Crt, i.e., of the wild-type (WT) β-subunit. Remarkably, the mutation of only two phenylalanine residues in the LH2 WT sequence, α-Phe at position − 12 and β-Phe at − 8, results in the loss of nearly 50% of functional Crt. Resonance Raman spectra indicate that the Crt conformation is fundamentally altered by the absence of the phenylalanines' aromatic side chains, suggesting that they lock the Crt into a precise, well-defined configuration. Thus, binding and specific functionalisation of Crt in the model and WT light-harvesting complex is reliant on the aromatic residue phenylalanine. The use of the light-harvesting complex as a model system thus substantiates the notion that the aromatic residue phenylalanine is a key factor for the binding of Crt to transmembrane proteins.     

Singlet-triplet fission of carotenoid excitation in light-harvesting LH2 complexes of purple phototrophic bacteria

The current generally accepted structure of light-harvesting LH2 complexes from purple phototrophic bacteria conflicts with the observation of singlet-triplet carotenoid excitation fission in these complexes. In LH2 complexes from the purple bacterium Allochromatium minutissimum, a drop in the efficiency of carotenoid triplet generation is demonstrated, which correlates with the extent of selective photooxidation of bacteriochlorophylls absorbing at ～850 nm. We conclude that singlet-triplet fission of carotenoid excitation proceeds with participation of these excitonically coupled bacteriochlorophylls. In the framework of the proposed mechanism, the contradiction between LH2 structure and photophysical properties of carotenoids is eliminated. The possibility of singlet-triplet excitation fission involving a third mediator molecule was not considered earlier.     

De-epoxidation of violaxanthin after reconstitution into different carotenoid binding sites of light-harvesting complex II

In higher plants, the de-epoxidation of violaxanthin (Vx) to antheraxanthin and zeaxanthin is required for the pH-dependent dissipation of excess light energy as heat and by that process plays an important role in the protection against photo-oxidative damage. The de-epoxidation reaction was investigated in an in vitro system using reconstituted light-harvesting complex II (LHCII) and a thylakoid raw extract enriched in the enzyme Vx de-epoxidase. Reconstitution of LHCII with varying carotenoids was performed to replace lutein and/or neoxanthin, which are bound to the native complex, by Vx. Recombinant LHCII containing either 2 lutein and 1 Vx or 1.6 Vx and 1.1 neoxanthin or 2.8 Vx per monomer were studied. Vx de-epoxidation was inducible for all complexes after the addition of Vx de-epoxidase but to different extents and with different kinetics in each complex. Analysis of the kinetics indicated that the three possible Vx binding sites have at least two, and perhaps three, specific rate constants for de-epoxidation. In particular, Vx bound to one of the two lutein binding sites of the native complex, most likely L1, was not at all or only at a slow rate convertible to Zx. In reisolated LHCII, newly formed Zx almost stoichiometrically replaced the transformed Vx, indicating that LHCII and Vx de-epoxidase stayed in close contact during the de-epoxidation reactions and that no release of carotenoids occurred.     

The incorporation of uracil into animal cell DNA in vitro

In the presence of dUTP, net DNA synthesis in vitro is substantially reduced. Small DNA fragments that arise during in vitro DNA synthesis in the presence of dUTP are produced as a result of dUMP incorporation and subsequent post-replication excision repair process initiated by uracil-DNA-glycosylase. The size of the fragments is dependent upon the amount of dUMP incorporated, but unlike the normal 4S intermediates of DNA synthesis, these repair products are not precursors to high molecular weight DNA but are further degraded. The high levels of dUTPase as well as the presence of RNA primers on most nascent DNA pieces (Tseng and Goulian, 1977) suggest that repair of uracil-containing DNA does not contribute to the generation of the small, nascent DNA pieces found during DNA synthesis in this in vitro system.     

Single-molecule spectroscopic characterization of light-harvesting 2 complexes reconstituted into model membranes

The spectroscopic properties of the light-harvesting 2 complexes (LH2) from the purple bacterium Rhodopseudomonas acidophila (strain 10050) in detergent micelles and reconstituted into lipid membranes have been studied by single-molecule spectroscopy. When LH2 complexes are solubilized from their host biological membranes by nondenaturing detergents, such as LDAO, there is a small 2-nm spectral shift of the B850 absorption band in the ensemble spectrum. This is reversed when the LH2 complexes are put back into phospholipid vesicles, i.e., into a more native-like environment. The spectroscopic properties on the single-molecule level of the detergent-solubilized LH2 complexes were compared with those reconstituted into the lipid membranes to see if their detailed spectroscopic behavior was influenced by these small changes in the position of the B850 absorption band. A detailed analysis of the low-temperature single-molecule fluorescence-excitation spectra of the LH2 complexes in these two different conditions showed no significant differences. In particular, the distribution of the spectral splitting between the circular k = +/-1 exciton states of the B850 absorption band and the distribution of the mutual angle between the k = +/-1 exciton states are identical in both cases. It can be concluded, therefore, that the LH2 complexes from Rps. acidophila are equally stable when solubilized in detergent micelles as they are when membrane reconstituted. Moreover, when they are solubilized in a suitable detergent and spin coated onto a surface for the single-molecule experiments they do not display any more structural disorder than when in a phospholipid membrane.     

The structure and function of bacterial light-harvesting complexes

The harvesting of solar radiation by purple photosynthetic bacteria is achieved by circular, integral membrane pigment-protein complexes. There are two main types of light-harvesting complex, termed LH2 and LH1, that function to absorb light energy and to transfer that energy rapidly and efficiently to the photochemical reaction centres where it is trapped. This mini-review describes our present understanding of the structure and function of the purple bacterial light-harvesting complexes.     

In vitro incorporation of tritium into native DNA

An exchange method is described for producing tritium-labeled native DNA in vitro with minimal physical damage to the DNA. Tritium-labeled calf thymus DNA prepared in this way has a specific activity of about 100 μCi/mmole of nucleotide (i.e., about 2 × 10⁸ dpm/mmole). Sedimentation velocity at neutral and alkaline pH indicate that the product has an average of two single strand breaks per duplex molecule of molecular weight 6 × 10⁶ daltons. The optical and thermal denaturation properties of the product are those of native DNA. The method should be particularly useful for labeling DNA from organisms that cannot be labeled conveniently in vivo.     

Refined carotenoid analysis of the major light-harvesting complex of Mantoniella squamata

The major light-harvesting complex (LHC) of the prasinophycean alga Mantoniella squamata is unique compared to other chlorophyll (Chl) a/b-binding LHC with respect to the primary protein structure and the pigmentation. Although the presence of Chl a, Chl b, a Chl c-type pigment and the xanthophylls neoxanthin, violaxanthin and prasinoxanthin was clearly determined, several carotenoids remained unidentified or were described controversially. We re-analysed the carotenoid composition and identified a new set of xanthophylls present in the LHC: uriolide, micromonol, micromonal and dihydrolutein. Additionally, one hydrophobic component was detected, presumably a xanthophyll. The pigment analysis in combination with quantitative protein determinations revealed a pigment-protein stoichiometry of 6 Chl a, 6 Chl b, 2 Chl c* and about 2 prasinoxanthin molecules per polypeptide. The other xanthophylls were present in sub-stoichiometric amounts. A comparison of results from LHC isolated either by sucrose density centrifugation or SDS-polyacryl gel electrophoresis revealed a decline in the amount of prasinoxanthin and a loss of violaxanthin using the latter preparation procedure, while the stoichiometric ratios of the other 6 xanthophylls remained constant. The fact that 8 different xanthophylls were found in the LHC of M. squamata can be explained best in terms of an oligomeric, presumably trimeric LHC organisation with subunits of heterogeneous pigmentation. Especially, the very stable assembly of most of the minor xanthophylls led to the assumption that these components play an important role in stabilisation and probably also in trunerisation of the LHC in vivo. This revised version was published online in August 2006 with corrections to the Cover Date.     

Reconstitution of light-harvesting complexes and photosystem II cores into galactolipid and phospholipid liposomes

Chlorophyll a/b light-harvesting complexes (chl a/b LHC) and photosystem II (PSII) cores were isolated from an octyl glucoside-containing sucrose gradient after solubilization of barley thylakoid membranes with Triton X-100 and octyl glucoside. No cation precipitation step was necessary to collect the chl a/b LHC. PAGE under mildly denaturing and fully denaturing conditions showed that the chl a/b LHC fraction contained chlorophyll-protein complexes CP27, CP29, and CP64. The PSII core material contained CP43 and CP47, and little contamination by other nonpigmented polypeptides. Freeze-fracture electron microscopy of the chl a/b LHC after reconstitution into digalactosyldiglyceride (DG) or phosphatidylcholine (PC) vesicles showed that the protein particles (approximately 7.5 +/- 1.6 nm) were approximately 99 and 90% randomly dispersed, respectively, in the liposomes. Addition of Mg++ produced particle aggregation and membrane adhesion in chl a/b LHC-DG liposomes in a manner analogous to that described for LHC-PC liposomes. Reconstitution of PSII cores into DG vesicles also produced proteoliposomes with randomly dispersed particles (approximately 7.5 +/- 1.6 nm). In contrast, PSII-PC mixtures formed convoluted networks of tubular membranes that exhibited very few fracture faces. Most of the protein particles (approximately 7.0 +/- 1.5 nm) were seen trapped between, rather than embedded in, the membranes. The interaction between the zwitterionic head group of the phosphatidyl choline and the negatively charged PSII core may be responsible for the unusual membrane structures observed.