Ultrastructure and freeze-fracture studies of the thylakoids ofMantoniella squamata (Prasinophyceae)

Summary The ultrastructure and the supramolecular organization of the thylakoids of the small green flagellate,Mantoniella squamata, were examined in thin sections and freeze-fracture preparations. The whole chloroplast is tightly packed with thylakoids, which show a pattern of meandering, branching and/or anastomosing membranes. In freeze-fracture preparations only two fracture-faces can be distinguished: the PF- and the EF-face. The PF-face has a much higher particle density than the EF-face (PF: 4086 particles/m²; EF: 865 particles/m²). The EF-face is not as uniform as the PF-face. The areas which are packed with particles probably correspond to closely appressed thylakoid regions or adhesive patches, noticed in thin sections in some areas. The mean particle size on both faces is also different (EF: 10.5 nm; PF: 8.6 nm), but no information about the classification of the particles to special protein complexes is available at this time.Abbreviations chl chlorophyll - EF exoplasmic fracture face - ER endoplasmic reticulum - LHC light-harvesting chlorophyll-protein complex - PF protoplasmic fracture face - PS I photosystem I - PS II photosystem II     

Occurrence of the putative microfibril-synthesizing complexes (linear terminal complexes) in the plasma membrane of the epiphytic marine red algaErythrocladia subintegra Rosenv.

Summary Cells of thalli at different developmental stages of the epiphytic marine red algaErythrocladia subintegra have been studied by freeze-etching. It was found that the plasma membrane exhibits linear microfibril-termnal synthesizing complexes (TCs), randomly distributed consisting of four rows of linearly-arranged particles (average diameter of particles 8.6 nm); each row of TCs consists of 5–33 particles (average 15). The TCs were observed on both fracture faces (PF and EF) but more clearly on the PF face. These structures appear to span both the outer and inner leaflets of the plasma membrane (transmembrane complexes)-The TCs have stable width (35 nm) and vary in length (41–311 nm, average 181 nm). The TCs subunits are highly ordered arrays forming a semicylinder. The average density of TCs on the PF face is 5.5TC/m². The microfibrils are randomly distributed and have a mean width of 39.4 nm (ranging from 16 to 70 nm). Many TCs are associated with the ends of microfibrils and microfibril imprints. The structural characteristics of linear TCs in the red algaErythrocladia are compared with those of the so far investigated Chlorophyta spp. All results favour the suggestion that TCs in the plasma membrane ofErythrocladia cells are involved in the biosynthesis, assembly and orientation of microfibrils.     

TIP CELL GROWTH AND THE FREQUENCY AND DISTRIBUTION OF CELLULOSE MICROFIBRIL-SYNTHESIZING COMPLEXES IN THE PLASMA MEMBRANE OF APICAL SHOOT CELLS OF THE RED ALGA PORPHYRA YEZOENSIS1

The supramolecular organization of the plasma membrane of apical cells in shoot filaments of the marine red alga Porphyra yezoensis Ueda (conchocelis stage) was studied in replicas of rapidly frozen and fractured cells. The protoplasmic fracture (PF) face of the plasma membrane exhibited both randomly distributed single particles (with a mean diameter of 9.2 ± 0.2 nm) and distinct linear cellulose microfibril-synthesizing terminal complexes (TCs) consisting of two or three rows of linearly arranged particles (average diameter of TC particles 9.4 plusmn; 0.3 nm). The density of the single particles of the PF face of the plasma membrane was 3000 μm^?2, whereas that of the exoplasmic fracture face was 325 μm^?2. TCs were observed only on the PF face. The highest density of TCs was at the apex of the cell (mean density 23.0 plusmn; 7.4 TCs μm^?2 within 5 μm from the tip) and decreased rapidly from the apex to the more basal regions of the cell, dropping to near zero at 20 μm. The number of particle subunits of TCs per μm² of the plasma membrane also decreased from the tip to the basal regions following the same gradient as that of the TC density. The length of TCs increased gradually from the tip (mean length 46.0 plusmn; 1.4 nm in the area at 0–5 μm from the tip) to the cell base (mean length 60.0 plusmn; 7.0 μm in the area at 15–20 μm). In the very tip region (0–4 μm from the apex), randomly distributed TCs but no microfibril imprints were observed, while in the region 4–9 μm from the tip microfibril imprints and TCs, both randomly distributed, occurred. Many TCs involved in the synthesis of cellulose microfibrils were associated with the ends of microfibril imprints. Our results indicate that TCs are involved in the biosynthesis, assembly, and orientation of cellulose microfibrils and that the frequency and distribution of TCs reflect tip growth (polar growth) in the apical shoot cell of Porphyra yezoensis. Polar distribution of linear TCs as “cellulose synthase” complexes within the plasma membrane of a tip cell was recorded for the first time in plants.     

Fusion of liposomes and chromatophores of Rhodopseudomonas capsulata: effect on photosynthetic energy transfer between B875 and reaction center complexes.

The photosynthetic chromatophore membranes of Rhodopseudomonas capsulata were fused with liposomes to investigate the effects of lipid dilution on energy transfer between the bacteriochlorophyll-protein complexes of this membrane. Phosphatidylcholine-containing liposomes were mixed with chromatophores at pH 6.0 to 6.2, and the mixture was fractionated on discontinuous sucrose gradients into four membrane fractions with lipid-to-protein ratios that varied 11-fold. Freeze-fracture electron microscopy revealed that the fractions contained closed vesicles formed by the fusion of liposomes to chromatophores. Particles with 9-nm diameters on the P fracture faces did not appear to change in size with increasing lipid content, but the number of particles per membrane area decreased proportionally with increases in the lipid-to-protein ratio. The bacteriochlorophyll-to-protein ratios, electrophoretic polypeptide profiles on sodium dodecyl sulfate-polyacrylamide gels, and light-induced absorbance changes at 595 nm caused by photosynthetic reaction centers were not altered by fusion. The relative fluorescence emission intensities due to the B875 light-harvesting complex increased significantly with increasing lipid content, but no increases in fluorescence due to the B800-B850 light-harvesting complex were observed. Electron transport rates, measured as succinate-cytochrome c reductase activities, decreased with increased lipid content. The results indicate an uncoupling of energy transfer between the B875 light-harvesting and reaction center complexes with lipid dilution of the chromatophore membrane.     

The role of enoyl-CoA hydratase in the metabolism of isoleucine byPseudomonas putida

Chromatium vinosum, strain D, exhibits two extreme modifications of near infra-red absorption spectra when growing heterotrophically at temperatures either above or below 36.5° C. Chromatophores isolated from cells grown either at 33° C (33° C chromatophores) or 39° C (39° C chromatophores) were analyzed for structural and functional parameters. For this the following chromatophore subunits were solubilized and characterized; (i) a fraction absorbing maximally at 800 nm with shoulders at 820 and 850 nm when derived from 33° C chromatophores or absorbing at 800 nm and 850 nm when derived from 39° C chromatophores; (ii) reaction center-light harvesting bacteriochlorophyll complexes with identical spectra and ratios of reaction center to light harvesting bacteriochlorophyll (1:45); (iii) complexes containing cytochromes, (IV) reaction center bacteriochlorophyll complexes. Irrespective of their origins the fractions exhibited qualitatively identical protein patterns as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.Protein patterns of 33° C and 39° C chromatophores revealed an identical ratio of proteins of reaction centers to proteins of cytochrome preparations. But the ratio of proteins of reaction centers to proteins of light harvesting moieties was 1.9 times higher in 39° C chromatophores than in 33° C chromatophores. Correspondingly, the ratio of reaction center per total bacteriochlorophyll was 1.7 times higher in 39° C chromatophores (1:110) then in 33° C chromatophores (1:190). Activities of photophosphorylation were 0.73 and 0.56 moles of ATP per moles of total bacteriochlorophyll per min for 33° C and 39° C chromatophores, respectively. Activities of sulfide oxidation in the light by whole cells were 2.37 and 1.96 moles of sulfide per mole of total bacteriochlorophyll per min for 33° C and 39° C cells. Accordingly, on a reaction center basis activities are significantly lower after growth of the organisms at 39° C than at 33° C. The data indicate that spectral changes in Chromatium vinosum represent changes in the ratio of reaction center to light harvesting bacteriochlorophyll accompanied by a variation of the absorption spectra of the latter bacteriochlorophyll moiety. Concomitantly, activities coupled to the photochemical apparatus were subjected to variations.Abbreviations Bchl Bacteriochlorophyll - LDAO lauryl dimethylamine oxide - SDS sodium dodecyl sulfate     

Organization of the cell membrane inEuglena

Summary The cell membrane ofEuglena gracilis has been investigated with the freeze-fracture technique. When split, this membrane produces two fracture faces which are striking in their non-complementarity. The P fracture face is covered with a high density of 110 Å (average diameter) particles, while the E face is made up of a complex series of striations occurring at regular angles to the pellicle ridges which encircle the organism. Under certain conditions, however, the structure of the P fracture face assumes a more ordered configuration, and striations are visible on this fracture face which are precisely complementary to those observed on the E face. These observations suggest that the cortical cell membrane ofEuglena may be organized along the lines of a two dimensional crystal. However, this pattern of organization is restricted to the cortical region of the cell membrane; as the membrane invaginates near the anterior end of the cell the fracture faces change abruptly, and organization more typical of other cell membranes is observed. This invagination forms an extensive reservoir in the anterior of the cell, and the membrane bounding it is distinctly fluid in structure, with clear examples of endo- and exocytosis observable. These differences suggest that the cell membrane inEuglena is divided into two distinct but contiguous regions, each specialized with regard to structure and function.     

Plasma-membrane rosettes in root hairs of Equisetum hyemale

Particle arrangement in the plasma membrane during cell wall formation was investigated by means of the double-replica technique in root hairs of Equisetum hyemale. Particle density in the protoplasmic fracture face of the plasma membrane was higher than in the extraplasmic fracture face. Apart from randomly distributed particles, particle rosettes were visible in the PF face of the plasma membrane. The rosettes consisted of six particles arranged in a circle and had an outer diameter of approx. 26 nm. No gradient in the number of rosettes was found, which agrees with micrifibril deposition taking place over the whole hair. The particle rosettes were found individually, which might indicate that they spin out thin microfibrils as found in higher-plant cell walls. Indeed microfibril width in these walls, measured in shadowed preparations, is 8.5±1.5 nm. It is suggested that the rosettes are involved in microfibril synthesis. Non-turgid cells lacked microfibril imprints in the plasma membrane and no particle rosettes were present on their PF face. Fixation with glutaraldehyde caused, probably as a result of plasmolysis, the microfibril imprints to disappear together with the particle rosettes. The PF face of the plasma membrane of non-turgid hairs sometimes showed domains in which the intramembrane particles were aggregated in a hexagonal pattern. Microfibril orientation during deposition will be discussed.Abbreviations EF extraplasmic fracture face - PF protoplasmic fracture face     

Regular arrays of intramembranous particles in the plasmalemma of guard cell and mesophyll cell protoplasts of Vicia faba

Freeze fractures of the plasmalemma membranes of guard-cell and mesophyll protoplasts of Vicia faba demonstrate that the inner monolayer of the plasmalemma is compartmentalized into areas with distinct, highly organized structures. Between areas of intramembranous particles dispersed randomly on a relatively smooth fracture face, membrane domains showing an extremely regular planar, hexagonal array of particles are interspersed. The dimensions of these hexagonal lattices are about 0.5 m in diameter, the center-to-center spacing is about 22 nm, and the particle size is about 9 nm. The particle in the hexagonal arrays are accompanied by complementary pits in the opposite monolayer fracture of the plasmalemma membrane.The freeze-fracture preparation was performed by using an improved Leybold Bioetch device which provides a sufficiently high cooling rate and allows the omission of cryoprotectants, like glycerol.Presented by H. Schnabl on the Workshop on Plant Membrane Transport, Toronto, Canada, July 1979     

Comparison, by freeze-fracture electron microscopy, of chromatophores, spheroplast-derived membrane vesicles, and whole cells of Rhodopseudomonas sphaeroides.

By using freeze-fracture electron microscopy, chromatophores and spheroplast-derived membrane vesicles from photosynthetically grown Rhodopseudomonas sphaeroides were compared with cytoplasmic membrane and intracellular vesicles of whole cells. In whole cells, the extracellular fracture faces of both cytoplasmic membrane and vesicles contained particles of 11-nm diameter at a density of about 5 particles per 10(4) nm2. The protoplasmic fracture faces contained particles of 11 to 12-nm diameter at a density of 14.6 particles per 10(4) nm2 on the cytoplasmic membrane and a density of 31.3 particles per 10(4) nm2 on the vesicle membranes. The spheroplast-derived membrane fraction consisted of large vesicles of irregular shape and varied size, often enclosing other vesicles. Sixty-six percent of the spheroplast-derived vesicles were oriented in the opposite way from the intracellular vesicle membranes of whole cells. Eighty percent of the total vesicle surface area that was exposed to the external medium (unenclosed vesicles) showed this opposite orientation. The chromatophore fractions contained spherical vesicles of uniform size approximately equal to the size of the vesicles in whole cells. The majority (79%) of the chromatophores purified on sucrose gradients were oriented in the same way as vesicles in whole cells, whereas after agarose filtration almost all (97%) were oriented in this way. Thus, on the basis of morphological criteria, most spheroplast-derived vesicles were oriented oppositely from most chromatophores.     

Particle density and protein composition of the peribacteroid membrane from soybean root nodules is affected by mutation in the microsymbiont Bradyrhizobium japonicum

Particle frequency of the peribacteroid membrane (PBM) from nodules of Glycine max (L.) Merr. cv. Maple Arrow infected with Bradyrhizobium japonicum 61-A-101 (wild-type strain) was determined by freeze-fracturing to be about 2200·m^-2 in the protoplasmic fracture face and 700·m^-2 in the exoplasmic fracture face. In membranes isolated from nodules infected with the mutant RH 31-Marburg of B. japonicum, the particle frequency was similar in both fracture faces with 1200–1300 particles·m^-2. Analysis of particlesize distribution on peribacteroid membranes showed a loss, especially of particle sizes larger than 11 nm, in the mutant-infected nodules. Two-dimensional gel electrophoresis (isoelectric focussing and sodium dodecyl sulfate-polyacrylamide) showed 27 different polypeptides in the PBM from nodules infected with the wild-type strain, four of which were absent from the PBM of nodules infected with the mutant RH 31-Marburg, which also exhibited one extra small-molecular-weight polypeptide. At least 14 of the 27 polypeptides in the PBM from the wild-type-infected nodule were glycoproteins. In three of these glycoproteins, post-translational modifications were either lacking or different when the membrane was derived from mutant-infected nodules.Abbreviations EF exoplasmatic fracture face - HRPO horse radish peroxidase - IEF Isoelectric focussing - PBM peribacteroid membrane - PF protoplasmatic fracture face - PNA peanut agglutinin - PSA Pisum sativum agglutinin - SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The assembly of cellulose microfibrils in Valonia macrophysa Kütz

The assembly of cellulose microfibrils was investigated in artificially induced protoplasts of the alga, Valonia macrophysa (Siphonocladales). Primary-wall microfibrills, formed within 72 h of protoplast induction, are randomly oriented. Secondary-wall lamellae, which are produced within 96 h after protoplast induction, have more than three orientations of highly ordered microfibrils. The innermost, recently deposited micofibrils are not parallel with the cortical microtubules, thus indicating a more indirect role of microtubules in the orientation of microfibrils. Fine filamentous structures with a periodicity of 5.0–5.5 nm and the dimensions of actin were observed adjacent to the plasma membrane. Linear cellulose-terminal synthesizing complexes (TCs) consisting of three rows, each with 30–40 particles, were observed not only on the E fracture (EF) but also on P fracture (PF) faces of the plasma membrane. The TC appears to span both faces of the bimolecular leaflet. The average length of the TC is 350 nm, and the number of TCs per unit area during primary-wall synthesis is 1 per m². Neither paired TCs nor granule bands characteristic of Oocystis were observed. Changes in TC structure and distribution during the conversion from primary- to secondary-wall formation have been described. Cellulose microfibril assembly in Valonia is discussed in relation to the process among other eukaryotic systems.Abbreviations TC terminal complex - EF E (outer leaflet) fracture face of the plasma membrane - PF P (inner leaflet) fracture face of the plasma membrane - MT microtubule - PS protoplasmic surface of the membrane     

Heliobacterium chlorum: cell organization and structure

The basic cellular organization of Heliobacterium chlorum is described using the freeze-etching technique. Internal cell membranes have not been observed in most cells, leading to the conclusion that the photosynthetic apparatus of these organisms must be localized in the cell membrane of the bacterium. The two fracture faces of the cell membrane are markedly different. The cytoplasmic (PF) face is covered with densely packed particles averaging 8 nm in diameter, while the exoplasmic (EF) face contains far fewer particles, averaging approximately 10 nm in diameter. Although a few differentiated regions were noted within these fracture faces, the overall appearance of the cell membrane was remarkably uniform. The Heliobacterium chlorum cell wall is a strikingly regular structure, composed of repeating subunits arranged in a rectangular pattern at a spacing of 11 nm in either direction. We have isolated cell wall fragments by brief sonication in distilled water, and visualized the cell wall structure by negative staining as well as deep-etching.Abbreviations PF protoplasmic fracture face - EF exoplasmic fracture face     

The plasma membrane of growing root hairs is composed of zones of local differentiation

Growing root hairs of cress (Lepidium sativum L.) were investigated using freeze-fracture and electron-microscopic techniques. Three zones of differentiation could be detected: the tip zone, the zone of vacuolation and the foot zone. Corresponding to these zones, the plasmatic fracture face of the plasma membrane showed areas of pronounced differentiation with respect to the distribution and frequency of intramembranous particles (IMPs). The tip zone was characterized by an irregular fracture plane caused by a large number of blisters which were more or less free of IMPs. These blisters coincided in size and shape with Golgi vesicles accumulated in the ground cytoplasm near the very tip. Outside these blisters, IMPs were randomly distributed. The surrounding cell wall was very thin and mainly composed of amorphous material. The plasma membrane of the vacuolation zone often revealed areas of hexagonally ordered particles (HOPS). Such patterns of particles were observed in chemically fixed and unfixed root hairs with a maximum surface density of 1200 HOPS per area. Mostly, however, 15–50 HOPS per area were found. The number of such areas increased with increasing distance from the tip up to five areas per m². Additionally, imprints of large cellulose microfibrils could be detected in unfixed material; they were mainly parallel to the root-hair axis and sometimes ended in areas of HOPS. However, HOPS were observed only in approximately 60% of the root hairs. Otherwise, large areas free of IMPs were interspersed between areas of randomly distributed IMPs. The particle frequency was relatively low and varied greatly in the tip as well as in the vacuolation zone, that is, from 1200 to 2000 IMPs m^-2. Finally, the plasma membrane of the foot zone showed a very constant number of approx. 2000 IMPs m^-2. These particles were mainly distinct and randomly distributed. In this zone, HOPS were never observed in spite of the fact that the cell wall was composed of numerous parallel-running cellulose microfibrils. Since membrane material is mainly incorporated in the tip zone where IMPs are statistically distributed, the results indicate that the plasma membrane of the outgrowing part of the root-hair cells is characterized by a high lateral mobility of its components. Furthermore, they indicate that specifically arranged particles are involved in the synthesis of cellulose microfibrils. These areas of HOPS seem to be locally restricted and — or limited with respect to their lifetime.Abbreviations cmf(s) cellulose microfibril(s) - EF extraplasmatic fracture face - HOPS hexagonally ordered particles - IMP intramembranous particle - PF plasmatic fracture face - pm plasma membrane Dedicated to Professor Dr. Kurt Mühlethaler, Zürich, on the occasion of his 65th birthday     

Insect UV-, and green-photoreceptor membranes studied by the freeze-fracture technique

Summary The membranes of the microvilli of UV- and green-photoreceptors of the ant Myrmecia gulosa have been studied with the freeze-fracture technique. Both inner fracture faces, the cytoplasmic P-face and the extracellular E-face, are covered by globular particles. The P-face particles appear to be randomly distributed, occasionally forming clusters. Their density is about 7,000/m², and their mean diameter is 8.5 nm. The E-face particles, however, are arranged in an ordered square pattern with a center-to-center spacing of 9 nm. The density and distribution of P- and E-face particles are the same in both the UV- and the green-photoreceptor membranes. No differences were found in the ultrastructural organization of photoreceptor membranes after dark or light adaptation. It is suggested that the P-face particles represent rhodopsin molecules.     

The plasma membrane of the Funaria caulonema tip cell: morphology and distribution of particle rosettes,and the kinetics of cellulose synthesis

Freeze-fracturing of Funaria hygrometrica caulonema cells leads to a cleavage within the plasma membrane. The extraplasmatic and the plasmatic fracture faces differ in their particle density. The plasmatic fracture face in caulonema tip cells or in tip cells of side branches, but never in other caulonema cells, is further characterized by the occurrence of particle rosettes. The highest density of rosettes is found at the cell apex but decreases steeply toward the cell base. The shape of the rosettes varies remarkably; 20% of them are found in an incomplete, presumably disintegrating or aggregating state. The complete rosette has a diameter of about 25 nm and consists of five to six particles. The size of the single particles varies between 4 nm to 10 nm. The rosettes are thought to posses cellulose-synthase activity. It is assumed that one rosette produces one elementary fibril; rough calculations, considering the number of rosettes and the estimated amount of cellulose produced in the tip region, indicate that an elementary fibrillar length of 900 nm is formed in 1 min by one rosette. The consequence of the kinetics on the life-time of the rosettes and the cellulose-synthase activity are discussed.Abbreviations EF extraplasmatic fracture face - PF plasmatic fracture face     

Ultrastructure of a cave-wall cyanophyte-Gloeocapsa NS4

Gloeocapsa strain NS4, a cyanophyte (cyanobacterium) which grows in low light levels inside cave entrances, was studied in the electron microscope by thin sectioning and freeze-etching. The cells are surrounded by a microfibrillar sheath divided by dense lamellae, which are probably an acidic mucopolysaccharide. Inside this is a typical Gramnegative cell wall. Double-replica freeze-fracture showed that the outer envelope of the wall fractures to give two faces each consisting of densely-packed particles; the particles of the outer leaflet seem to consist of subunits arranged in a hollow cylinder. A structural model of the outer envelope is proposed. The plasma membrane fractures to give a PF face with 3000 9 nm particles m^-1 and an EF face with 150–700 11–12 nm particles m^-1. The thylakoids are arranged in a pattern not previously found in a unicellular cyanophyte, parallel arrays which intersect, and may fuse with, the plasma membrane. The thylakoid membranes have 2,850 particles m^-1, mean size 10.9 nm, on the PF face and 560 particles m^-1, mean size 12.3 nm, on the EF face. Phycobilisomes are difficult to see, but may be unusually large. These ultrastructural features may be adaptations to a very low light habitat.     

Properties of photochemical reaction centers purified from Rhodopseudomonas gelatinosa

Reaction centers were isolated from a carotenoidless mutant of Rhodopseudomonas gelatinosa by hydroxyapatite chromatography of purified chromatophores treated with lauryl dimethyl amine oxide. Absorption spectra and spectra of light-induced absorbance changes are similar to those of reaction centers from Rhodopseudomonas sphaeroides. The ratio of absorbance at 280 nm to that at 799 nm was 1.8 in the purest preparations. The extinction coefficient at the 799 nm absorption maximum was estimated to be 305 ± 20 mM^?1 · cm^?1. The molecular weight based on protein and chromophore assays was found to be 1.5 · 10⁵; the reaction center protein accounted for 6% of the total membrane protein. These reaction centers contained no cytochrome and showed just two components of apparent molecular weights 33 000 and 25 000 in polyacrylamide gel electrophoresis. The chromatophores contained 42 molecules of antenna bacteriochlorophyll for each reaction center.     

Pigment biogenesis in freshwater shrimp ventral nerve chord chromatophores

Summary The possible biogenesis of two pigment granule types present in the monochromatic, brown chromatosomes enveloping the ventral nerve chord of the freshwater palaemonid shrimps Macrobrachium acanthurus, M. heterochirus and M. olfersii is examined by transmission electron microscopy in thin section and freeze fracture replicas. Prominent, membrane limited granules are suggested to have their origin in a complex, juxtanuclear, smooth endoplasmic reticulum labyrinth, continuous with the nuclear envelope. Amembranous, lipocarotenoid granules possibly derive from the external surface of the smooth endoplasmic reticulum. Nuclear envelope and SER membranes contain numerous 11 nm diameter intramembranous particles while pigment granule membranes exhibit fewer particles. A dictyosomal origin for the lipocarotenoid granules is discounted. Granulogenesis is suggested to be a continuous process in crustacean chromatophores.     

The IleL229 → Met mutation impairs the quinone binding to the QB-pocket in reaction centers of Rhodobacter sphaeroides

A spontaneous mutant (R/89) of photosynthetic purple bacterium Rhodobacter sphaeroides R-26 was selected for resistance to 200 M atrazin. It showed increased resistance to interquinone electron transfer inhibitors of o-phenanthroline (resistance factor, RF=20) in UQ_o reconstituted isolated reaction centers and terbutryne in reaction centers (RF=55) and in chromatophores (RF=85). The amino acid sequence of the Q_B binding protein of the photosynthetic reaction center (the L subunit) was determined by sequencing the corresponding pufL gene and a single mutation was found (Ile^L229 Met). The changed amino acid of the mutant strain is in van der Waals contact with the secondary quinone Q_B. The binding and redox properties of Q_B in the mutant were characterized by kinetic (charge recombination) and multiple turnover (cytochrome oxidation and semiquinone oscillation) assays of the reaction center. The free energy for stabilization of Q_AQ_B ^– with respect to Q_A ^–Q_B was G_AB=–60 meV and 0 meV in reaction centers and G_AB=–85 meV and –46 meV in chromatophores of R-26 and R/89 strains at pH 8, respectively. The dissociation constants of the quinone UQ_o and semiquinone UQ_o ^– in reaction centers from R-26 and R/89 showed significant and different pH dependence. The observed changes in binding and redox properties of quinones are interpreted in terms of differential effects (electrostatics and mesomerism) of mutation on the oxidized and reduced states of Q_B.Abbreviations BChl bacteriochlorophyll - Ile isoleucine - Met methionin - P primary donor - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - RC reaction center protein - UQ_o 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50 This work is dedicated to the memory of Randall Ross Stein (1954–1994) and is, in a small way, a testament to the impact which Randy's ideas have had on the development of the field of competitive herbicide binding.