Isolation and characterization of light harvesting bacteriochlorophyll · protein complexes from Rhodopseudomonas capsulata

The isolation of two native light harvesting bacteriochlorophyl · protein complexes from Rhodopseudomonas capsulata is described. The light harvesting bacteriochlorophyll I (B 875) has been isolated from the blue-green mutant Ala⁺ lacking both carotenoids and light harvesting bacteriochlorophyll II. Light harvesting bacteriochlorophyll I is associated with a protein (light harvesting band 2) of 12 000 molecular weight.Light harvesting bacteriochlorophyll II complex has been isolated from the mutant Y5 lacking a reaction center and light harvesting bacteriochlorophyll I. Light harvesting bacteriochlorphyll II (B 800 + 850) together with carotenoids is associated with two polypeptides (light harvesting bands 3 and 4) having molecular weights of about 8000 and 10 000 (sodium dodecyl sulfate polyacrylamide gel electrophoresis). A third protein (light harvesting band 1) is in the purified light harvesting II fraction (mol. wt. approx. 14 000), but not associated with bacteriochlorophyll or carotenoids. The amino acid composition of the 3 antenna pigment II proteins is given. The polarity of these proteins was found to be 48%. From the amino acid composition the following molecular weights were calculated band 1: 17 350, band 3: 13 350 and band 4: 10 500.     

The formation of bacteriochlorophyll · protein complexes of the photosynthetic apparatus of Rhodopseudomonas capsulata during early stages of development

Cells of Rhodopseudomonas capsulata cultivated at an oxygen partial pressure of 400 mmHg in the dark contained 0.1 nmol or less total bacteriochlorophyll per mg membrane protein. The bacteriochlorophyll was found in the reaction center (10 pmol bacteriochlorophyll/mg membrane protein) and in the light harvesting bacteriochlorophyll I but not in the light harvesting bacteriochlorophyll II. Formation of the photosynthetic apparatus in those cells was induced by incubation at a very low oxygen tension in the dark. Reaction center bacteriochlorophyll and light harvesting bacteriochlorophyll increased three fold after 60 min of incubation at 1–2 mmHg (pO₂). Light harvesting bacteriochlorophyll II increased strongly after 60 min and became dominating after 90 min of incubation. The total bacteriochlorophyll content doubled every 30 min, but synthesis of reaction center bacteriochlorophyll proceeded at much lower rates. Consequently the size of the photosynthetic unit (total bacteriochlorophyll/reaction center bacteriochlorophyll) increased from 15 to 52 during 150 min of incubation. The proteins of the photosynthetic apparatus were synthesized concomitantly with bacteriochlorophyll.Cells which were incubated at 0.5 mmHg (pO₂) do not grow but form the photosynthetic apparatus. During the first hours of incubation light harvesting bacteriochlorophyll I and reaction center bacteriochlorophyll were the dominant bacteriochlorophyll species, but light harvesting bacteriochlorophyll II was synthesized only in small amounts. Total bacteriochlorophyll and reaction center bacteriochlorophyll increased from 30 min up until 210 min of incubation more than 10 fold. The final concentrations of total bacteriochlorophyll and reaction center bacteriochlorophyll were 8.6 nmol and 0.26 nmol per mg membrane protein, respectively. The three protein components of the reaction centers (mol. wts. 28 000, 24 000 and 21 000) and the protein of the light harvesting I complex (mol. wt. 12 000) were incorporated simultaneously. The protein of band 1 (mol. wt. 14 000) which was present in the isolated light harvesting complex II, was synthesized only in very small amounts. The proteins of bands 3 and 4 (mol. wt. 10 000 and 8000) however, which were shown to be associated with light harvesting bacteriochlorophyll II, were synthesized in noticeable amounts as was light harvesting bacteriochlorophyll II. In addition a protein with an apparent molecular weight of 45 000 showed a strong incorporation of ¹⁴C-labeled amino acids. This protein comigrates with one protein which was found to be associated with a green pigment excreted during incubation at 0.5 Torr into the medium. The in vivo-absorption maxima of this pigment complex were 660, 590, 540, 417 and 400 nm. The succinate oxidase and the NADH oxidase seemed to be incorporated into the newly formed intracytoplasmic membrane only in very small amounts. Thus, reaction center and light harvesting bacteriochlorophyll and their associated proteins were simultaneously synthesized, whereas light harvesting complex II is the variable part of the photosynthetic apparatus.     

Relations between pigments and proteins in the photosynthetic membranes of Rhodopseudomonas spheroides

We have isolated from Rhodopseudomonas spheroides a pigment-protein complex of apparent weight 9 kdaltons that bears more than 60% of the light harvesting bacteriochlorophyll. The isolation procedure involved exposure to 1% lauryl dimethyl amine oxide (LDAO). The purified 9-kdalton fraction showed the light harvesting bacteriochlorophyll components B800 and B850, plus carotenoids. The ratio of bacteriochlorophyll to protein was 17%. This protein is probably the same as the “band 15” protein of Fraker and Kaplan. It may exist in vivo as characteristic aggregates of higher molecular weight. LDAO added to Rps. spheroides chromatophores converted the bacteriochlorophyll component B870 to a form absorbing at 770 nm but had little effect on the “B800 + B850” system, causing only a reversible shift of the 850-nm band to 845 nm. Anti-reaction center serum, added to subcellular fractions from Rps. spheroides with 1% LDAO, precipitated reaction center chromoprotein unaccompanied by light harvesting bacteriocholorophyll. Other antisera precipitated light harvesting components and left the reaction center chromophores in solution. A major protein of apparent weight 45 kdaltons was found in relatively nonpigmented fractions from Rps. spheroides, associated with cell wall fragments. The 45-kdalton protein showed considerable interstrain variability, whereas the 9-kdalton and reaction center proteins appeared constant.     

Isolation and Characterization of a Proteolipid in Defatted Soybean Meals

A proteolipid was isolated from the chloroform–methanol (2:1, by vol.) extract of defatted soybean meals by a modified Folch method. The proteolipid gave a yield of 0.05% of the defatted meals, and the ratio of protein and lipid was neary 3:4. The complex gave a single band containing both protein and lipid on polyacrylamide gel electrophoresis. TLC analysis of the lipid moiety showed that the major components were glycolipids and phospholipids. The protein moiety contained more hydrophobic amino acids and less acidic amino acids in comparison with the amino acid composition of soybean globulin. The protein moiety contained two kinds of protein component (I and II) which have molecular weights of 13,000 (I) and 15,000 (II) on SDS-urea polyacrylamide gel electrophoresis, and N-terminal amino acids of alanine (I) and glutamic acid (II). The apoprotein is a new protein and different from the whey proteins or globulins of soybean.     

The protein patterns of intracytoplasmic membranes and reaction center particles isolated fromRhodopseudomonas capsulata

Intracytoplasmic membranes of wild type strain 37 b 4 and mutant strains A1a car^-bchl^-, A1a car^-bchl⁺ ofRhodopseudomonas capsulata were isolated. The membrane proteins were solubilized and separated by polyacrylamide gel electrophoresis (methods of Takayamaet al., 1964; Weber and Osborn, 1969). The band patterns were compared with each other. From the strain A1a car^-bchl⁺ reaction center particles were isolated by treatment of membrane with Triton X-100 followed by sucrose density gradient centrifugation. The reaction center particles were found to be enriched in reaction center bacteriochlorophyll. This pigment shows a reversible bleaching at 855 nm and a blue shift at 798 nm. The light harvesting bacteriochlorophyll portion of this fraction was 14–22% of the total bacteriochlorophyll content. The three main proteins of the reaction center particles amount to about 80% of the total protein of the particles. The molecular weights of the main proteins were estimated to be 32000, 27500 and 22500 daltons.     

Comparative study of B890 pigment-lipoprotein complexes from sulfur (Chromatium minutissimum) and non-sulfur (Rhodopseudomonas palustris) purple photosynthesizing bacteria]

Pigment-lipoprotein B890 complexes containing reaction center and "light-focusing" bacteriochlorophyll a were isolated from photosynthetic membranes of sulfur (Chromatium minutissimum) and non-sulfur (Rhodopseudomonas palustris) purple bacteria after the treatment with Triton X-100. The molecular weights of complexes were evaluated using several methods (200 000-300 000). By means of electron microscopy the sizes of complexes were found to be about 80 A. On the air-water interface hexagonal packing of complexes was observed. The chemical compositions of complexes are very similar except bacteriochlorophyll a whose specific content is somewhat higher in Chromatium minutissimum. The protein composition of complexes was studied and the molecular weights of proteins were estimated by SDS-gel electrophoresis. The results obtained show significant similarities in molecular organization of B890 complexes isolated from sulfur (Chromatium minutissimum) and non-sulfur (Rhodopseudomonas palustris) purple bacteria.     

Function of membrane proteins coupled to bacteriochlorophyll synthesis: Studies with wild type and mutant strains of Rhodopseudomonas spheroides

Membrane proteins with estimated molecular weights of 26,000, 22,000, 19,000, and 10,000–5,000 (designated 9, 10, 11, and 15, respectively) were found previously to be coupled to the synthesis of bacteriochlorophyll in Rhodopseudomonas spheroides. They have been attributed to the reaction center complex (proteins 9, 10, 11) and light-harvesting forms (protein 15) on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane fractions from the wild type and by analysis of certain photosynthetic mutants. Three of the mutants form light harvesting but not reaction-center bacteriochlorophyll; their membranes lack proteins 10 and 11, though proteins 9 and 15 are detectable. These mutants have little or no photophosphorylation or light-induced transhydrogenase activities and their respiration is not inhibited by illumination. Another mutant, strain 71-20 apparently has functional reaction centers, as determined by the above criteria, yet it does not grow anaerobically in the light for unknown reasons.     

Identification of subunits of bovine heart cytochrome oxidase.

Purified lipid-depleted cytochrome oxidase, at purity of 12--14 nmol heme a per mg protein, has been shown to contain seven non-identical subunits in the ratio of unity. Their molucular weights on polyacrylamide gel are, in thousands, 40, 21, 14.8, 13.5, 11.6, 9.5, and 7.6 from gel electrophoresis after dissociation in sodium dodecyl sulfate and beta-mercaptoethanol. The molar ratio is determined by the amino acid composition of each subunit obtained from direct hydrolysis of the stained polyacrylamide gel slices. The amino acid composition of the isolated subunits I and II determined by regular hydrolysis method is found practically the same as that from direct hydrolysis of gel slices. The heme-associated polypeptides are identified with subunits of molecular weights of 40.10(3) and 11.6.10(3). One of the two coppers associated with the polypeptide of molecular weight of 21 000. The second copper may be associated with heme in the subunit of 40.10(3). Evidence of the existence of interpolypeptide disulfide linkages is presented.     

Studies on cytochrome c oxidase, I. Purification and characterization of bovine myocardial enzyme and identification of peptide chains in the complex]

As part of the preliminary work for the structural elucidation of cytochrome c oxidase, the enzyme complex was isolated from bovine heart muscle and characterised chemically. The enzyme contains 10-11 nmol haem a, and 12-13 nmol copper per mg protein. The solubilised active enzyme also contains 5% phospholipid, comprising about 2 mol each of cardiolipin and phosphatidylethanolamine per mol haem a. In addition, the preparation contains a small number of detergent molecules (Tween-80). Eight polypeptide components were isolated by preparative dodecylsulphate gel electrophoresis, gel filtration on Biogel P-60, and counter current distribution. The apparent molecular weights of these components were I - 36 000, II - 28 000 (21 000), III - 19 000, IV - 14 000, V - 12 500, VI - 11 000, VII - 10 000 and VIII - 6000. At least seven intact polypeptide chains contribute to the structure of the enzyme complex of the terminal oxidase. On the basis of amino acid analysis and end group determination, they can be divided into two groups. The high molecular weight peptides I -III are hydrophobic and their amino acid compositions differ markedly from those of known enzyme proteins, especially with respect to their contents of leucine and methionine. Components I and II have formyl methionine at their N-termini. They are therefore possibly mitochondrial membrane components from complex 4 of the respiratory chain. Polypeptides IV - VII resemble functional enzyme subunits in their amino acid composition. Some of them possess free N-termini (alanine). The low molecular weight component VIII is heterogeneous and contains the N-terminal amino acids isoleucine, serine and phenylelanine in non-stoichiometric amounts. Analysis gives a minimal protein molecular weight of 130 000 (65 000 per haem a) for the two haem and two copper-containing "monomers". The molecular weight of the moiety preliminarily defined as enzymatic is about 48 000. The chemical characterisation provides data for the strategy of the subsequent sequence analysis of the polypeptides.     

Function of a PscD subunit in a homodimeric reaction center complex of the photosynthetic green sulfur bacterium Chlorobium tepidum studied by insertional gene inactivation. Regulation of energy transfer and ferredoxin-mediated NADP+ reduction on the cytoplasmic side

The PscD subunit in the homodimeric "type I" photosynthetic reaction center (RC) complex of the green sulfur bacterium Chlorobium tepidum was disrupted by insertional mutagenesis of its relevant pscD gene. This is the first report on the use of the direct mutagenic approach into the RC-related genes in green sulfur bacteria. The RC complex of C. tepidum is supposed to form a homodimer of two identical PscA subunits together with three other subunits: PscB (FA/FB-containing protein), PscC (cytochrome cz), and PscD. PscD shows a relatively low but significant similarity in its amino acid sequence to PsaD in the photosystem I of plants and cyanobacteria. We studied the biochemical and spectroscopic properties of a mutant lacking PscD in order to elucidate its unknown function. 1) The RC complex isolated from the mutant cells showed no band corresponding to PscD on SDS-PAGE analysis. 2) The growth rate of the PscD-less mutant was slower than that of the wild-type cells at low light intensities. 3) Time-resolved fluorescence spectra at 77 K revealed prolonged decay times of the fluorescence from bacteriochlorophyll c on the antenna chlorosome and from bacteriochlorophyll a on the Fenna-Matthews-Olson antenna protein in the mutant cells. The loss of PscD led to a much slower energy transfer from the antenna pigments to the special pair bacteriochlorophyll a (P840). 4) The mutant strain exhibited slightly less activity of ferredoxin-mediated NADP+ photoreduction compared with that in the wild-type strain. The extent of suppression, however, was less significant than that reported in the PsaD-less mutants of cyanobacterial photosystem I. The evolutionary relationship between PscD and PsaD was also discussed based on a structural homology modeling of the former.     

Studies on the apoproteins of the major lipoprotein of the yolk of hen's eggs. I. Isolation and properties of the low-molecular-weight apoproteins.

In a continuing study of protein-lipid interactions in egg yolk, the total apoprotein mixture (i.e. the 'apovitellenins') from the high-lipid, low-density lipoprotein (density 0.97 g/ml) of the yolk from hen's eggs has been isolated in a soluble form. By gel-filtration chromatography in 6M urea the mixture has been separated into several fractions from which three new low-molecular-weight proteins (I, Ia, and II), making up about 30% of the total, have been isolated. The most plentiful of these (I) consists of stable aggregates with several identical subunits each of molecular weight about 10 000. This protein is analogous to the principal protein from the corresponding lipoprotein of emu's egg yolk, i.e. emu's apovitellenin I. Hen's apovitellenin I has a slightly different amino acid composition from that of the emu; notably it contains a sulphydryl group. The hen's protein also forms more stable aggregates that are dissociated by detergent and by guanidine hydrochloride but are stable in urea. The molecular weight of Ia is similar to that of I and the amino acid composition is the same, with the exception that Ia has a higher proportion of amide groups. It aggregates less readily than I under the same conditions. The third new protein (II, 'hens's apovitellenin II') has a molecular weight of about 20 000. It has no tyrosine or methionine residues, but contains glucosamine and has several disulphide groups. It has been isolated in very small amount only.     

Dimeric nature and amino acid compositions of homogeneous canine prostatic human liver and rat liver acid phosphatase isoenzymes. Specificity and pH-dependence of the canine enzyme.

Two isoenzymes of rat liver acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) have been purified to homogeneity, at least one of these for the first time. Both of the rat liver isoenzymes have identical specific activities towards p-nitrophenyl phosphate. Molecular weights of the native enzymes are 92 000 for rat liver isoenzyme I and 93 000 for isoenzyme II, while the subunit molecular weights are 51 000 and 52 000 respectively. Data on substrate specificity and pH dependence are presented for the homogeneous canine prostatic enzyme, which is also isolated as a dimeric enzyme of (native) molecular weight 89 000. Carbohydrate analysis data are presented for canine prostatic acid phosphatase and it is further noted that both isoenzymes of rat liver acid phosphatase are also glycoproteins. The amino acid compositions of the two rat liver isoenzymes are presented together with those of the similar dimeric acid phosphatase of human liver and of canine prostate. Comparison of these results with published data for the amino acid composition of human prostatic acid phosphatase shows substantial similarities. However, significant differences are seen in the amino acid composition of rat liver acid phosphatase isoenzyme I as compared to a previous literature report. Most notably, 17 histidine residues are found per mol of isoenzyme I and 18 for isoenzyme II.     

The consequences of chlorophyll deficiency for photosynthetic light use efficiency in a single nuclear gene mutation of cowpea

The light harvesting and photosynthetic characteristics of a chlorophyll-deficient mutant of cowpea (Vigna unguilata), resulting from a single nuclear gene mutation, are examined. The 40% reduction in total chlorophyll content per leaf area in the mutant is associated with a 55% reduction in pigment-proteins of the light harvesting complex associated with Photosystem II (LHC II), and to a lesser extent (35%) in the light harvesting complex associated with Photosystem I (LHC I). No significant differences were found in the Photosystem I (PS I) and Photosystem II (PS II) contents per leaf area of the mutant compared to the wildtype parent. The decreases in the PS I and PS II antennae sizes in the mutant were not accompanied by any major changes in quantum efficiencies of PS I and PS II in leaves at non-saturating light levels for CO₂ assimilation. Although the chlorophyll deficiency resulted in an 11% decrease in light absorption by mutant leaves, their maximum quantum yield and light saturated rate of CO₂ assimilation were similar to those of wildtype leaves. Consequently, the large and different decreases in the antennae of PS II and PS I in the mutant are not associated with any loss of light use efficiency in photosynthesis.Abbreviations LHC I, LHC II light harvesting chlorophyll a/b protein complexes associated with PS I and PS II - A₈₂₀ light-induced absorbance change at 820 nm - ø_{PS I}, ø_{PS II} relative quantum efficiencies of PS I and PS II photochemistry     

Low molecular weight angiotensin I converting enzyme from rat lung.

A low molecular weight angiotensin I converting enzyme (light angiotensin enzyme) was isolated from a homogenate of rat lung subjected to dialysis against sodium acetate at pH 4.8. This enzyme has a molecular weight of 84 000 on Sephadex G-200 and a molecular weight of 91 000 on SDS-poly-acrylamide gel as compared with a molecular weight of 139 000 for angiotensin I converting enzyme on SDS-polyacrylamide. Light angiotensin enzyme was activated by NaCl and inhibited by EDTA, angiotensin II, and bradykinin potentiating factor nonapeptide. Light angiotensin enzyme cross-reacted with antibody prepared against angiotensin I converting enzyme and stained with periodic acid-Schiff reagent as a glycoprotein. The evidence suggests that light angiotensin enzyme is a fragment of the higher molecular weight enzyme.     

Evolution and functional properties of photosystem II light harvesting complexes in eukaryotes

Photoautotrophic organisms, the major agent of inorganic carbon fixation into biomass, convert light energy into chemical energy. The first step of photosynthesis consists of the absorption of solar energy by pigments binding protein complexes named photosystems. Within photosystems, a family of proteins called Light Harvesting Complexes (LHC), responsible for light harvesting and energy transfer to reaction centers, has evolved along with eukaryotic organisms. Besides light absorption, these proteins catalyze photoprotective reactions which allowed functioning of oxygenic photosynthetic machinery in the increasingly oxidant environment. In this work we review current knowledge of LHC proteins serving Photosystem II. Balance between light harvesting and photoprotection is critical in Photosystem II, due to the lower quantum efficiency as compared to Photosystem I. In particular, we focus on the role of each antenna complex in light harvesting, energy transfer, scavenging of reactive oxygen species, chlorophyll triplet quenching and thermal dissipation of excess energy. This article is part of a Special Issue entitled: Photosystem II.     

Structure of cytochromec oxidase from baker's yeast—a progress report. Preparation of four subunits for amino acid sequence determination and attempts to localize the cytochromec binding site

Summary Cytochromec oxidase from the inner membrane of yeast mitochondria consists of seven nonidentical protein subunits, three being synthesized on mitochondrial ribosomes (molecular weights I: 43 K, II: 34 K, and III: 24 K) and four being made on cytoplasmic ribosomes (molecular weights IV: 14 K, V: 12 K, VI: 12 K, and VII: 4.5 K).In the present study all four cytoplasmically synthesized subunits of the enzyme were isolated on a large scale using ion exchange chromatography and gel filtartion. Their amino acid composition as well as their amino- and carboxy-terminal amino acid residues have been determined. Sequence determinations of sub-units IV and VI are already in an advanced state. The sequence of subunit VI is characterized by a large amino-terminal stretch dominated by charged amino acid residues followed by a cluster of hydrophobic amino acids.The binding site of yeast cytochrome oxidase for cytochromec was studied by chemical crosslinking experiments. The formation of a disulfide bridge between the two proteins was observed by using cytochromec from yeast modified with 5-thionitrobenzoate at the cysteinyl residue in position 107. Alternatively, a disulfide between yeast cytochromec and the oxidase could be formed directly by oxidation with copper phenanthroline. Gel electrophoresis of the crosslinked complexes in sodium dodecyl sulfate revealed a new protein band with an apparent molecular weight of 38 K. This new band appears to be derived from cytochromec and from subunit III of cytochrome oxidase.Recipient of a fellowship from the Swiss National Science Foundation. Present address: Department of Biology, University of California at San Diego, La Jolla, Calif. 92037 (USA).     

Effects of light intensity on membrane differentiation in Rhodopseudomonas capsulata.

Cells of Rhodopseudomonas capsulata, strain 37b4, leu-, precultivated anaerobically under low light intensity, were exposed to high light intensity (2000 W.m-2). The cells grew with a mass doubling time of 3 h. The synthesis of bacteriochlorophyll (BChl) began after two doublings of cell mass. Reaction center and light-harvesting BChl I (B-875) were the main constituents of the photosynthetic apparatus incorporated into the membrane. The size of the photosynthetic unit (total BChl/reaction center) decreased and light-harvesting BChl I became the dominating BChl species. Concomitant with the appearance of the different spectral forms of BChl the respective proteins were incorporated into the membrane, i.e. the three reaction center polypeptides, the polypeptide associated with light-harvesting BChl I, the two polypeptides associated with BChl II. A polypeptide of an apparent molecular weight of 45 000 was also incorporated. A lowering of the light intensity to 7 W.m-2 resulted in a lag phase of growth for 6 h. Afterwards, the time for doubling of cell mass was 11 h. The concentration of all three BChl complexes (reaction center, light-harvesting BChl I and II complexes)/cell and per membrane protein increased immediately. Also the size of the photosynthetic unit and the amount of intracytoplasmic membranes/cell increased. The activities of photophosphorylation, succinate dehydrogenase, NADH dehydrogenase and NADH oxidation (respiratory chain)/membrane protein are higher in membrane preparations isolated from cells grown at high light intensities than in such preparations from cells grown at low light intensities.     

Modulator protein as a component of the myosin light chain kinase from chicken gizzard.

The Ca2+-dependent regulation of smooth muscle actomyosin involves a myosin light chain kinase (ATP: myosin light chain phosphotransferase). It has been shown (Dabrowska, R., Aromatorio, D., Sherry, J.M.F., and Hartshorne, D.J. 1977, Biochem. Biophys. Res. Commun. 78, 1263) that the kinase is composed of two proteins of approximate molecular weights 105 000 and 17 000. In this communication it is demonstrated that the 17 000 component is the modulator protein. This conclusion is based on: (1) the identical behavior of the 17 000 kinase component and modulator protein in assays of actomyosin Mg2+-ATPase activity, phosphorylation of myosin, and phosphodiesterase activity, and, (2) the similarity of the 17 000 kinase component and the modulator protein with respect to amino acid composition, absorption spectrum, and electrophoresis in urea-polyacrylamide gels. It is shown also that the modulator protein from smooth muscle and troponin C are distinct proteins.     

Comparison of extracellular ATP and UTP signalling in rat renal mesangial cells. No indications for the involvement of separate purino- and pyrimidino-ceptors.

The visible c.d. spectrum of wild-type Rhodospirillum rubrum shows positive bands [Dratz, Schultz & Sauer (1966) Brookhaven Symp. Biol. 19, 303-318] that are largely due to the B880 antenna pigments, bacteriochlorophyll a and carotenoids. The bacteriochlorophyll c.d. band was absent from the spectrum of R. rubrum G9, a mutant unable to synthesize coloured carotenoids, and could be partly restored by adding extracted carotenoids to freeze-dried membrane vesicles isolated from that mutant. Therefore it seems to arise from either bacteriochlorophyll-carotenoid interactions or bacteriochlorophyll-protein interactions that are induced by the carotenoid. The more complex carotenoid c.d. band had different shapes in native and reconstituted carotenoid-containing membranes. Such differences suggest that the optical activity of the carotenoid in the B880 antenna arises from both non-degenerate and degenerate interactions.