Thermophilic Cyanobacterium Mastigocladus Laminosus Research Articles

Genomics of variation in nitrogen fixation activity in a population of the thermophilic cyanobacterium Mastigocladus laminosus.

Variation in phenotypic traits that contribute to fitness influences a population's evolutionary response and its impact on ecosystem function following environmental change, yet its amount and nature are rarely known. Here, we investigated variation in nitrogen (N) fixation activity and its genetic basis for a random sample of laboratory strains of the cyanobacterium Mastigocladus laminosus from a N-limited, geothermally influenced stream in Yellowstone National Park. In a linear mixed-effects model, temperature and genetic differences among strains were the most important factors explaining variation in activity. Genome-wide analyses of genetic divergence between groups of strains that varied in N fixation activity revealed that few loci were strongly associated with these phenotypic differences. Notably, a single nonsynonymous polymorphism in the sulfate assimilation gene apsK explained >25% of the variation in activity at high temperature. We further identified a role for allelic variation of multiple terminal cytochrome oxidases for different aspects of N fixation. In addition, genomes of strains that fixed the most N overall contained a nonsense mutation in a histidine kinase gene that is expected to disrupt normal protein function and may result in transcriptional rewiring. This study illustrates how taking complementary approaches to link phenotype and genotype can inform our understanding of microbial population diversity.

The Iron-Sulfur Cluster of the Rieske Iron-Sulfur Protein Functions as a Proton-exiting Gate in the Cytochrome bc1 Complex

The destruction of the Rieske iron-sulfur cluster ([2Fe-2S]) in the bc(1) complex by hematoporphyrin-promoted photoinactivation resulted in the complex becoming proton-permeable. To study further the role of this [2Fe-2S] cluster in proton translocation of the bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with mutations at the histidine ligands of the [2Fe-2S] cluster were generated and characterized. These mutants lacked the [2Fe-2S] cluster and possessed no bc(1) activity. When the mutant complex was co-inlaid in phospholipid vesicles with intact bovine mitochondrial bc(1) complex or cytochrome c oxidase, the proton ejection, normally observed in intact reductase or oxidase vesicles during the oxidation of their corresponding substrates, disappeared. This indicated the creation of a proton-leaking channel in the mutant complex, whose [2Fe-2S] cluster was lacking. Insertion of the bc(1) complex lacking the head domain of the Rieske iron-sulfur protein, removed by thermolysin digestion, into PL vesicles together with mitochondrial bc(1) complex also rendered the vesicles proton-permeable. Addition of the excess purified head domain of the Rieske iron-sulfur protein partially restored the proton-pumping activity. These results indicated that elimination of the [2Fe-2S] cluster in mutant bc(1) complexes opened up an otherwise closed proton channel within the bc(1) complex. It was speculated that in the normal catalytic cycle of the bc(1) complex, the [2Fe-2S] cluster may function as a proton-exiting gate.

Structural Basis for the Thermostability of Ferredoxin from the Cyanobacterium Mastigocladus laminosus

Plant-type ferredoxins (Fds) carry a single [2Fe-2S] cluster and serve as electron acceptors of photosystem I (PSI). The ferredoxin from the thermophilic cyanobacterium Mastigocladus laminosus displays optimal activity at 65 degrees C. In order to reveal the molecular factors that confer thermostability, the crystal structure of M.laminosus Fd (mFd) was determined to 1.25 A resolution and subsequently analyzed in comparison with four similar plant-type mesophilic ferredoxins. The topologies of the plant-type ferredoxins are similar, yet two structural determinants were identified that may account for differences in thermostability, a salt bridge network in the C-terminal region, and the flexible L1,2 loop that increases hydrophobic accessible surface area. These conclusions were verified by three mutations, i.e. substitution of L1,2 into a rigid beta-turn ((Delta)L1,2) and two point mutations (E90S and E96S) that disrupt the salt bridge network at the C-terminal region. All three mutants have shown reduced electron transfer (ET) capabilities and [2Fe-2S] stability at high temperatures in comparison to the wild-type mFd. The results have also provided new insights into the involvement of the L1,2 loop in the Fd interactions with its electron donor, the PSI complex.

The Inhibitor DBMIB Provides Insight into the Functional Architecture of the QoSite in the Cytochromeb6fComplex

Previously [Roberts, A. G., and Kramer, D. M. (2001) Biochemistry 40, 13407-13412], we showed that 2 equiv of the quinone analogue 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) could occupy the Q(o) site of the cytochrome (cyt) b(6)f complex simultaneously. In this work, a study of electron paramagnetic resonance (EPR) spectra from the oriented cyt b(6)f complex shows that the Rieske iron-sulfur protein (ISP) is in distinct orientations, depending on the stoichiometry of the inhibitor at the Q(o) site. With a single DBMIB at the Q(o) site, the ISP is oriented with the 2Fe-2S cluster toward cyt f, which is similar to the orientation of the ISP in the X-ray crystal structure of the cyt b(6)f complex from thermophilic cyanobacterium Mastigocladus laminosus in the presence of DBMIB, as well as that of the chicken mitochondrial cyt bc(1) complex in the presence of the class II inhibitor myxothiazol, which binds in the so-called "proximal niche", near the cyt b(L) heme. These data suggest that the high-affinity DBMIB site is at the proximal niche Q(o) pocket. With >or=2 equiv of DBMIB bound, the Rieske ISP is in a position that resembles the ISP(B) position of the chicken mitochondrial cyt bc(1) complex in the presence of stigmatellin and the Chlamydomonas reinhardtii cyt b(6)f complex in the presence of tridecylstigmatellin (TDS), which suggests that the low-affinity DBMIB site is at the distal niche. The close interaction of DBMIB bound at the distal niche with the ISP induced the well-known effects on the 2Fe-2S EPR spectrum and redox potential. To further test the effects of DBMIB on the ISP, the extents of cyt f oxidation after flash excitation in the presence of photosystem II inhibitor DCMU were measured as a function of DBMIB concentration in thylakoids. Addition of DBMIB concentrations at which a single binding was expected did not markedly affect the extent of cyt f oxidation, whereas higher concentrations, at which double occupancy was expected, increased the extent of cyt f oxidation to levels similar to that of cyt f oxidation in the presence of a saturating concentration of stigmatellin. Simulations of the EPR g-tensor orientations of the 2Fe-2S cluster versus the physical orientations based on single-crystal studies of the cyt bc(1) complex suggest that the soluble ISP domain of the spinach cyt b(6)f complex can rotate by at least 53 degrees, which is consistent with long-range ISP domain movement. Implications of these results are discussed in the context of the X-ray crystal structures of the chicken mitochondrial cyt bc(1) complex and the M. laminosus and C. reinhardtii cyt b(6)f complexes.

Evolution of photosynthesis: time-independent structure of the cytochrome b6f complex.

Structures of the cytochrome b(6)f complex obtained from the thermophilic cyanobacterium Mastigocladus laminosus and the green alga Chlamydomonas reinhardtii, whose appearance in evolution is separated by 10(9) years, are almost identical. Two monomers with a molecular weight of 110,000, containing eight subunits and seven natural prosthetic groups, are separated by a large lipid-containing "quinone exchange cavity". A unique heme, heme x, that is five-coordinated and high-spin, with no strong field ligand, occupies a position close to intramembrane heme b(n). This position is filled by the n-side bound quinone, Q(n), in the cytochrome bc(1) complex of the mitochondrial respiratory chain. The structure and position of heme x suggest that it could function in ferredoxin-dependent cyclic electron transport as well as being an intermediate in a quinone cycle mechanism for electron and proton transfer. The significant differences between the cyanobacterial and algal structures are as follows. (i) On the n-side, a plastoquinone molecule is present in the quinone exchange cavity in the cyanobacterial complex, and a sulfolipid is bound in the algal complex at a position corresponding to a synthetic DOPC lipid molecule in the cyanobacterial complex. (ii) On the p-side, in both complexes a quinone analogue inhibitor, TDS, passes through a portal that separates the large cavity from a niche containing the Fe(2)S(2) cluster. However, in the cyanobacterial complex, TDS is in an orientation that is the opposite of its position in the algal structure and bc(1) complexes, so its headgroup in the M. laminosus structure is 20 A from the Fe(2)S(2) cluster.

Purification and crystallization of the cytochrome b6f complex in oxygenic photosynthesis.

The cytochrome b6f complex from the thermophilic cyanobacterium Mastigocladus laminosus and spinach chloroplasts has been purified as a dimeric species. It was found by electrospray ionization mass spectroscopy to contain eight and nine subunits, respectively, and dimeric masses of 217,070 and 286,454 Da. The subunits common to the complex from both sources are petA (cytochrome f), B (cytochrome b6), C (Rieske iron-sulfur protein), D (subunit IV), and small 3.2-4.2 kDa polypeptides petG,L,M, and N. The ninth polypeptide, the 35 kDa petH poly-peptide in the spinach complex, was identified as ferredoxin NADP reductase (FNR), which binds to the complex tightly at a stoichiometry of approx 0.9 (cyt f)-1. The spinach complex contains diaphorase activity diagnostic of FNR, and is active in facilitating ferredoxin-dependent electron transfer from NADPH to the cytochrome b6f complex. The purified cytochrome b6f complex contains stoichiometrically bound chlorophyll a and beta-carotene at a ratio of one per cytochrome f, and bound lipid, in which MGDG and PG are the most abundant species. The delipidated highly purified complexes are active immediately after preparation and for approx 1 wk if left on ice, transferring 300-350 electrons/cyt f/s. Both complexes are subject to proteolysis and associated loss of activity if left for extended periods (>1 wk) at room temperature. Addition of pure synthetic lipid to the delipidated M. laminosus complex (the "lipid augmentation" technique) allows rapid and ready formation of large (>0.2 mm) crystals suitable for x-ray diffraction analysis and structure determination, which diffract with good statistics to 3.0 A.

Purification, crystallization and preliminary X-ray analysis of ferredoxin isolated from thermophilic cyanobacterium Mastigocladus laminosus.

Ferredoxins are soluble iron-sulfur proteins that are involved in numerous electron-transfer reactions. Plant-type ferredoxins, which carry a single [2Fe-2S] cluster, serve as the electron acceptors of Photosystem I. The ferredoxin from the thermophilic cyanobacterium Mastigocladus laminosus has unique thermostable properties. The isolated protein is active at temperatures of higher than 338 K. The gene encoding the ferredoxin from M. laminosus was subcloned into an expression vector and overproduced in Escherichia coli. The recombinant protein was purified to near-homogeneity and crystallized using the hanging-drop method. Thin needle-like crystals were grown in several crystallization conditions but were unsuitable for X-ray analysis owing to poor scattering. In order to obtain better diffracting crystals, ferredoxin was purified directly from M. laminosus cells. Single crystals were obtained using 30% PEG 4000, 0.32 M Mg(NO(3))(2), 20 mM Tris-HCl pH 8.2. These crystals diffracted to 1.25 A resolution using synchrotron radiation and were found to belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 28.45, b = 50.93, c = 110.91 A. The asymmetric unit was found to contain two ferredoxin molecules, with a corresponding V(M) of 1.9 A(3) Da(-1) and a solvent content of 34%. The present study indicates that overcoming the poor diffraction of crystals obtained from recombinant protein can be achieved by producing crystals from protein purified from the native host.

Capsular polysaccharide produced by the thermophilic cyanobacterium Mastigocladus laminosus. Structural study of an undecasaccharide obtained by lithium degradation.

The capsular polysaccharide produced by the thermophilic cyanobacterium Mastigocladus laminosus has been subjected to a specific degradation with lithium in ethylenediamine. The released undecasaccharide attached to one unit of tetrahydroxycyclopentanecarboxylic acid has been characterized by a combination of 2D nuclear magnetic resonance spectroscopy, mass spectrometry, monosaccharidic composition and linkage analyses. From the overlap of the structure of this oligosaccharide with previously identified di-, tri- and pentasaccharides released by mild acid hydrolysis, the capsular polysaccharide was deduced to have a pentadecasaccharide repeating unit with the following structure:

Structural features and assembly of the soluble overexpressed PsaD subunit of photosystem I

PsaD is a peripheral protein on the reducing side of photosystem I (PS I). We expressed the psaD gene from the thermophilic cyanobacterium Mastigocladus laminosus in Escherichia coli and obtained a soluble protein with a polyhistidine tag at the carboxyl terminus. The soluble PsaD protein was purified by Ni-affinity chromatography and had a mass of 16716 Da by MALDI-TOF. The N-terminal amino acid sequence of the overexpressed PsaD matched the N-terminal sequence of the native PsaD from M. laminosus. The soluble PsaD could assemble into the PsaD-less PS I. As determined by isothermal titration calorimetry, PsaD bound to PS I with 1.0 binding site per PS I, the binding constant of 7.7×10 6 M −1, and the enthalpy change of −93.6 kJ mol −1. This is the first time that the binding constant and binding heat have been determined in the assembly of any photosynthetic membrane protein. To identify the surface-exposed domains, purified PS I complexes and overexpressed PsaD were treated with N-hydroxysuccinimidobiotin (NHS-biotin) and biotin-maleimide, and the biotinylated residues were mapped. The Cys 66, Lys 21, Arg 118 and Arg 119 residues were exposed on the surface of soluble PsaD whereas the Lys 129 and Lys 131 residues were not exposed on the surface. Consistent with the X-ray crystallographic studies on PS I, circular dichroism spectroscopy revealed that PsaD contains a small proportion of α-helical conformation.

Structural characterization of three aldobiuronic acids derived from the capsular polysaccharide produced by the thermophilic cyanobacterium Mastigocladus laminosus

This study deals with the chemical characterization of a capsular polysaccharide (CPS) produced by a thermal biomass formed by the cyanobacterium Mastigocladus laminosus. Acid hydrolysis performed on the purified polysaccharide has led to the isolation of several acid-resistant oligosaccharides. Two of them have already been reported and assigned as: α− GlcA−(1→2)−α− GalA−(1→2)− Man. and α− GlcA−(1→2)−α− GalA−(1→2)−β− Man−(1→4)−β− Gal(1→2)− Rha . In this report, results on the isolation and partial purification of three supplementary oligosaccharidic units are presented. Gas chromatography-mass spectrometry (GC-MS) and H-nuclear magnetic resonance (NMR) spectroscopy investigations allowed them to be assigned as three aldobiuronic acids with the following structures: α− GlcA−(1→3)− Gal α− GlcA−(1→3)− Fucα− GalA−(1→3)− Fuc

Molecular assembly of the phycobilisomes from the cyanobacterium Mastigocladus laminosus

Five phycocyanin- and two phycoerythrocyanin-associated linker polypeptides were resolved by two-dimensional gel electrophoresis from isolated phycobilisomes of the thermophilic cyanobacterium Mastigocladus laminosus. These linker polypeptides were located in the substructure of the phycobilisome by analysis of isolated high molecular weight allophycocyanin—phycocyanin (AP—PC) complexes and by examination of different phycobilisome “types” induced by alteration of the culture conditions. The core fractions reveal two different rod—core linker polypeptides L RC 29.5PC and L RC 32PC and one rod linker L R 31PC associating the first and the following “trimeric” PC complexes at the profile sides of the core. The “minimal” phycobilisome type with an apparent molecular weight M r of 4 500 000 is characterized by the occurrence of only one additional rod linker polypeptide L R 34.5PC. Further sequential elongation of the rods occurs by PC and phycoerythrocyanin (PEC) “trimers” in cooperation with two low molecular weight linker polypeptides L R 11PC and L R 11PEC. The structure of the “maximal” phycobilisome with M r 8 500 000 is completed by the association of PEC “hexamers” via L R 34.5PEC. This sequential assembly could be demonstrated by the polypeptide composition of different “intermediate” and “maximal” phycobilisomes. The large number of linker polypeptides and the stoichiometric calculations of core fractions and of different phycobilisome “types” indicate a partial “trimeric” organization of the rod complexes. With respect to polypeptide stoichiometry, the “trimeric” arrangement of PC·L RC 29.5/32, PC·L R 31, PC·L R 11 and PEC·L R 11 in the structure of the rods is proposed. The structural and functional consequences of the study are represented in a model of the “maximal” phycobilisome.

Monomeric and trimeric forms of photosystem I reaction center of Mastigocladus laminosus: crystallization and preliminary characterization.

Photosystem I (PSI) reaction centers (RCs) of the thermophilic cyanobacterium Mastigocladus laminosus were purified and characterized. The PSI RC was obtained in two forms, monomeric and trimeric. The two forms contained the same number of pigments per P700 and displayed similar photochemical activities. The two forms had nearly identical polypeptide subunit compositions; the only observed difference was an additional subunit of about 12 kDa observed in the trimeric form. The purified preparations of both the monomeric and the trimeric forms were used for crystallization and preliminary crystallographic analysis. The trimeric PSI RC preparations produced several three-dimensional crystal forms, one of which, the "hexagonal needle" form (THN), had a hexagonal unit cell with dimensions of 300 x 300 x 160 A, containing four PSI RC trimers. The monomeric preparations also produced single crystals of several forms under various crystallization conditions. One of these crystal forms, the "hexagonal plate" (MHP), diffracted to a resolution of about 5.5 A. It had a hexagonal unit cell with dimensions of 192 x 192 x 163 A, containing six PSI RC monomers. Comparison of the PSI RCs in the crystals with those in the precrystallization preparations demonstrated that neither the monomeric nor the trimeric form of PSI RC was altered by the crystallization process. Both forms retained their original polypeptide subunit composition and their pigment content.

Morphological and physiological characteristics of a non-heterocystous strain of the cyanobacterium Mastigocladus laminosus Cohn from fumarolic soil on Mt Erebus, Antarctica

An unusual strain of the thermophilic cyanobacterium Mastigocladus laminosus occurs on warm soils on the volcano Mt Erebus (77°32′S, 167°8′E). It differs morphologically from the two genetically distinct forms described from thermal habitats elsewhere. Heterocysts are lacking and true-branching is rare. Its photosynthetic rate, and the contrasting rates of two less thermotolerant algae from Mt Erebus soils, Phormidium fragile (Cyanobacteria) and Pseudococcomyxa simplex (Chlorophyta), were measured over the range -2° to 62°C. The optimum temperature range of M. laminosus was 35° to 50°C. Photosynthetic response to temperature of all three algae in the laboratory correlated well with distribution patterns in the field, confirming that zonation patterns were temperature controlled. M. laminosus retained viability following exposure to deep-freezing, freeze-thaw cycles and desiccation. Viability of the alga in culture was lost following exposure to 50°C in darkness for 42 days and following 42 days in the light at 0°C. Discussion suggests the alga would survive long distance airborne dispersal in the desiccated condition but would not survive the duration of overwinter darkness on moist soils at the warmer end of its range of occurrence in the field.

Refined three-dimensional structure of phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus at 2.7 Å

The structure of the phycobiliprotein phycoerythrocyanin from the thermophilic cyanobacterium Mastigocladus laminosus has been determined at 2.7 Å resolution by X-ray diffraction methods on the basis of the molecular model of C-phycocyanin from the same organism. Hexagonal phycoerythrocyanin crystals of space group P6 3 with cell constants a = b = 156.86 A ̊ , c = 40.39 A ̊ , α = β = 90 °, γ = 120 ° are almost isomorphous to C-phycocyanin crystals. The crystal structure has been refined by energy-restrained crystallographic refinement and model building. The conventional crystallographic R-factor of the final model was 19.2% with data to 2.7 Å resolution. In phycoerythrocyanin, the three (αβ)-subunits are arranged around a 3-fold symmetry axis, as in C-phycocyanin. The two structures are very similar. After superposition, the 162 C α atoms of the α-subunit have a mean difference of 0.71 Å and the 171 C α atoms of the β-subunit differ by 0.51 Å. The stereochemistry of the chiral atoms in the phycobiliviolin chromophore A84 is C (31)-R, C (4)-S. The configuration of the chromophore is C (10)-Z, C (15)-Z and the conformation C (5)- anti, C (9)- syn and C (14)- anti like the phycocyanobilin chromophores in phycoerythrocyanin and C-phycocyanin.

Preparation and characterization of Photosystem II particles from a thermophilic cyanobacterium

Photosystem II particles have been isolated and purified from the thermophilic cyanobacterium Mastigocladus laminosus after sulfobetaine treatment. The light-induced steady-state electron transport, flash-induced absorption changes at 830 nm and 325 nm, reflecting the turnover of P-680 and of the acceptor side, respectively, have been measured. In the presence of artificial electron acceptors the preparations showed high O 2-evolution activities (e.g., 3000–4000 μmol O 2 per mg Chl a per h with phenyl- p-benzoquinone) which were DCMU-sensitive. O 2-evolution activity was stable in the presence of various cations (10 mM) and of glycerol (25% (v/v) in the suspending medium. Furthermore, the activity was stable up to 60°C and maximum rate of the electron transport was observed at 50°C. The spectrum of the particles showed a chlorophyll a maximum at approx. 673 nm, some carotenoids were still present but phycocyanin was completely removed. After SDS-polyacrylamide gel electrophoresis the preparations contained mainly nine protein bands at 110, 105, 60, 55, 35, 33, 22, 18 and 17 kDa. Under repetitive laser flash excitation the P-680 +-reduction kinetics contained components in the nanosecond and microsecond range with an absorption rate of approx. 1:1. When PS-II particles were purified at room temperature by anion-exchange chromatography nanosecond relaxation kinetics were observed even though the particles did not evolve oxygen. The gel analysis for this preparation showed mainly four protein bands at 60, 55, 35 and 18 kDa.

Allophycocyanin complexes of the phycobilisome from Mastigocladus laminosus. Influence of the linker polypeptide L8.9C on the spectral properties of the phycobiliprotein subunits.

The following phycobiliproteins and complexes of the allophycocyanin core were isolated from phycobilisomes of the thermophilic cyanobacterium Mastigocladus laminosus: alpha AP, beta AP, (alpha AP beta AP), (alpha AP beta AP)3, (alpha AP beta AP)3L8.9C, (alpha APB alpha AP2 beta AP3)L8.9C. The six proteins and complexes were characterised spectroscopically with respect to absorption, oscillator strength, extinction coefficient, fluorescence emission, relative quantum yield, fluorescence emission polarisation and fluorescence excitation polarisation. The interpretation of the spectral data was based on the three-dimensional structure model of (alpha PC beta PC)3 (Schirmer et al. (1985) J. Mol. Biol. 184, 257-277), which is related to the allophycocyanin trimer. The absorption and CD spectra of the complexes (alpha AP beta AP)3, (alpha AP beta AP)3L8.9C and (alpha APB alpha AP2 beta AP3)L8.9C could be deconvoluted into the spectra of the phycobiliprotein subunits. The assumptions made for the deconvolution could be checked by the synthesis of the spectra of (alpha APB beta AP)3. The synthesised spectra are in good agreement with the corresponding measured spectra published by other authors. Considering the deconvoluted spectra the following influences on the chromophores could be ascribed to L8.9C: L8.9C neither influences the alpha AP nor the alpha APB chromophores. L8.9C shifts the absorption maximum of the beta AP chromophore to longer wavelength than the absorption maximum of the alpha AP chromophore in trimeric complexes. L8.9C increases the oszillator strength of the beta AP chromophores to about the value of the alpha AP chromophores in trimeric complexes. L8.9C turns the beta AP chromophores from sensitizing into weak fluorescing chromophores. By means of the hydropathy plot and the predicted secondary structure, a postulated three-fold symmetry in the tertiary structure of L8.9C could be confirmed.

Functional assignment of chromophores and energy transfer in C phycocyanin isolated from the thermophilic cyanobacterium Mastigocladus laminosus

The optical characteristics and pathway of energy transfer in the C phycocyanin trimer isolated from the thermophilic cyanobacterium Mastigocladus laminosus were investigated at steady state by absorption, circular dichroism, fluorescence and fluorescence polarization spectroscopy. Based on the comparison of optical data with the 3-dimensional structure of the C-phycocyanin trimer determined by X-ray analysis (Schirmer, T., Bode, W., Huber, R., Sidler, W. and Zuber, H. (1984) in Proceedings of the Symposium on Optical Properties and Structure of Tetrapyrroles, (Blauer, G. and Sund, M., eds.), pp. 445–449, Walter de Gruyter, Berlin, and (1985) J. Mol. Biol. 184, 257–277), the functional assignment of three types of chromophore was established. An α subunit has an s chromophore and the chromophores at the positions 84 and 155 in the amino acid sequence of the β subunit are assigned as f and s chromophores, respectively. In the C phycocyanin trimer energy transfer occurs from the α chromophore in one monomer to the β f chromophore in an adjacent monomer, and from the β s chromophore to the β f chromophore in the same monomer. The direction of energy flow is from the outside to the inside of the trimer, where the locus for the binding of a colourless polypeptide is postulated. In the phycobilisomes the energy concentrated at the β f chromophores might be transferred toward the allophycocyanin core mainly by the β f chromophores in the phycocyanin rods.

X-ray crystallographic structure of the light-harvesting biliprotein C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus and its resemblance to globin structures

The structure of the biliprotein C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus has been determined at 3 Å resolution by X-ray diffraction methods. Phases have been obtained by the multiple isomorphous replacement method. The electron density map could be improved by solvent flattening and has been interpreted in terms of the amino acid sequence. The protein consists of three identical (αβ)-units which are arranged around a threefold symmetry axis to form a disc of approximate dimensions 110 Å × 30 Å with a central channel of 35 Å in diameter. This aggregation form is supposed to be the same as that found in the rods of native phycobilisomes. Both subunits, α and β, exhibit a similar structure and are related by a local twofold rotational axis. Each subunit is folded into eight helices and irregular loops. Six helices are arranged to form a globular part, whereas two helices stick out and mediate extensive contact between the subunits. The arrangement of the helices of the globular part resembles the globin fold: 59 equivalent C α-atoms have a root-mean-square deviation of 2.9 Å. The chromophores attached to cystein 84 of the α- and β-subunits are topologically equivalent to the haem. All three chromophores of C-phycocyanin, open-chain tetrapyrroles, are in an extended conformation. α84 and β84 are attached to helix E (globin nomenclature), β155 is linked to the G-H loop. The shortest centre-to-centre distance between chromophores in the trimer is 22 Å.

Respiration in energy-transducing membranes of the thermophilic cyanobacterium Mastigocladus laminosus: I. Relation of the respiratory and photosynthetic electron transport

The thermophilic cyanobacterium Mastigocladus laminosus was grown at different CO 2 concentrations and temperatures. Respiratory and photosynthetic electron transport in isolated membranes were measured and their activities were compared. Cells grown at low CO 2 concentration showed respiratory electron transport, whereas Photosystem-II-dependent transport was optimal in cells grown at high CO 2 concentrations. The respiratory electron transport from NADH and succinate were KCN-sensitive, whereas NADPH-dependent O 2 uptake was not. It could be shown that NADH and succinate donate electrons in the photosynthetic electron pathway via Photosystem I. In cytochrome- c-553-depleted membranes added cytochrome c-553 could stimulate photosynthetic and respiratory electron transport. A common electron transport pathway between the quinone and cytochrome c is postulated.

Respiration in energy-transducing membranes of the thermophilic cyanobacterium Mastigocladus laminosus: II. Oxidative phosphorylation

Oxidative phosphorylation was measured in isolated energy-transducing membranes of the thermophilic cyanobacterium Mastigocladus laminosus with NADH-mediated electron transport. This dark phosphorylation was similar to photophosphorylation in its sensitivity to uncouplers and energy-transfer inhibitors. However, photophosphorylation was 20- to 50-times more active than oxidative phosphorylation. The P O ratio of oxidative phosphorylation was about 0.2. Besides oxidative phosphorylation, adenylate kinase- and ADP-P i exchange activity were measured in the dark. The ADP-P i exchange reaction was identified as polynucleotide phosphorylase.