Glycolate excretion by Rhodospirillum rubrum

Glycolate can be measured in the supernatant fraction after incubation of butyrate-grown cells of Rhodospirillum rubrum either colorimetrically by the Calkins method or enzymatically using glycolate oxidase. Under optimal conditions, half-maximal excretion occurs at 11% O₂ and the maximal rate is 6.9 nmol of glycolate min^-1 mg protein^-1 at 30°C. The pH and temperature optima are 7.6 and 30°C and light intensity is saturating in the range of 2–10×10⁴ lux. Carbon dioxide inhibits glycolate excretion and exogenous butyrate stimulates. Glycolate excretion is maximal by butyrate-light grown cells harvested in the early stationary phase and under all conditions is proportional to the cellular content of ribulose 1,5-bisphosphate carboxylase/oxygenase.Non-Standard Abbreviations Bicine (N,N-bis[2-hydroxyethyl]glycine) - RuBP d-ribulose-1,5-bisphosphate - HPMS 2-pyridylhydroxymethanesulfonate     

The Structure of Rhodospirillum rubrum

Cells from serial cultures of R. rubrum, grown anaerobically in the light, were harvested at intervals from ½ to 15 days and sectioned for electron microscopy by conventional methods. Cells of this species possess a multilayered outer envelope, and the external cell surface is differentiated into ridges extending parallel or obliquely to the long axis of the cell. Cells from very young cultures resemble non-photosynthetic bacteria and contain only a granular cytoplasm, scattered high-density particles, and low-density areas corresponding to the chromatin areas observed by light microscopy. They contain neither the chromatophores nor the lamellar systems assumed by previous investigators to be characteristic of this species when grown anaerobically in the light. Chromatophores appear in cells from cultures older than about 12 hours, while systems of paired lamellae appear along with the chromatophores in cells from cultures older than about 8 days. Divergent opinions concerning the occurrence of chromatophores or lamellae in this species can be resolved on the basis of the age of cultures used in previous studies. Other changes occurring in cells from cultures of increasing age include the appearance of granular and reticulate cytoplasmic bodies and vacuoles, extension of the chromatin areas, and the appearance of a single membrane enclosing several chromatophores.     

Glutathione reductase from Rhodospirillum rubrum

Summary Glutathione reductase (NADPH¹: glutathione oxidoreductase (EC 1.6.4.2) was purified 70 fold from Rhodospirillum rubrum by ammonium sulfate fractionation, gelfiltration with Sephadex and chromatography on DEAE-cellulose. The optimum pH of the reaction is 7.5–8.2 K _m values of 8.4×10^–6 M for NADPH and 5.8×10^–5 M for GSSG were determined. The kinetic data indicate a bisubstrate reaction mechanism. The prosthetic group is FAD (K _m 1.1×10^–6M). The flavin can be completely dissociated from the enzyme, and 70% of the original activity can subsequently be restored by FAD. The molecular weight was determined with a calibrated column Sephadex G-200 and found to be approximately 63,000. The enzyme is inhibited reversibly by several anions. With iodide the inhibition is competitive with respect to GSSG. Sulfhydryl reagents (N-ethylmaleinimide, p-chlormercuribenzoate) strongly inhibit the enzyme when it is present in the reduced state. The enzyme is reduced by low concentrations of NADPH and by higher concentrations of NADH. GSSG protects the enzyme against this inhibition. The enzyme is reversibly inhibited by incubation with NADPH or NADH.

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Zusammenfassung Glutathionreduktase wurde aus Rhodospirillum rubrum mit Ammoniumsulfatfraktionierung, Gelfiltration mit Sephadex und Chromatographie an DEAE-Cellulose 70 fach angereichert. Das pH Optimum der Reaktion liegt bei 7,5–8,2. K _m -Werte: 8,4·10^–6 M für NADPH und 5,8·10^–5 M für GSSG. Aus den kinetischen Daten ergibt sich für das Enzym ein Bisubstratreaktionsmechanismus. Die prosthetische Gruppe ist FAD (K _m 1,1·10^–6 M). Das Flavin kann vollständig vom Enzymprotein abdissoziiert werden, durch erneute Zugabe von FAD können etwa 70% der ursprünglichen Aktivität zurückerhalten werden. Das Molekulargewicht, bestimmt durch Gelfiltration mit einer kalibrierten Säule Sephadex G-200, ist ca. 63000. Das Enzym wird durch verschiedene Anionen reversibel gehemmt. Bei J^– ist die Hemmung kompetitiv mit GSSG. Sulfhydrylreagentien (N-Äthylmaleinimid und p-Chlomercuribenzoat) sind potente Inhibitoren, wenn das Enzym im reduzierten Zustand vorliegt. Das Enzym kann bereits durch niedrige Konzentrationen an NADPH sowie durch höhere Konzentrationen an NADH reduziert werden. GSSG schützt das Enzymprotein gegen die Hemmung durch Sulfhydryl-reagentien. Das Enzym wird durch Inkubation mit NADPH und NADH reversibel gehemmt.

     

Untersuchungen zur Photophosphorylierung bei Rhodospirillum molischianum und Rhodospirillum rubrum

Zusammenfassung Die Thylakoide aus Rhodospirillum rubrum und Rhodospirillum molischianum werden nach Homogenisation der Zellen mit Ultraschall durch fraktionierte Zentrifugation isoliert. An diese Membranstrukturen ist das System der Photophosphorylierung gebunden. Die Aktivität dieses Systems in Abhängigkeit vom Redoxpotential des Mediums wird untersucht. Die stärkste Bindung anorganischen Phosphates wird unter Edelgasatmosphäre bei Zusatz von Spuren eines Elektronendonators (0,07 mol Succinat je Ansatz) beobachtet. Die cyclische Photophosphorylierung wird einerseits durch Sauerstoff und oxydierende Verbindungen wie K₃Fe(CN)₆ anderseits durch Überreduktion mittels reduzierter Redoxverbindungen wie 2,6-Dichlorphenolindophenol oder Phenazinmethosulfat (beide reduziert durch Ascorbat) unter Wasserstoffatmosphäre gehemmt. Die Sauerstoffhemmung kann durch reduziertes Phenazinmethosulfat zu 50% aufgehoben werden. Antimycin A blockiert die lichtabhängige Phosphorylierung; 2,4-Dinitrophenol dagegen hemmt kaum. Die zellfreien Systeme beider Arten zeigen die gleiche Abhängigkeit vom Redoxpotential obwohl R. rubrum wesentlich sauerstofftoleranter ist als R. molischianum und auch durch oxydative Phosphorylierung im Dunkeln ATP bilden kann. Die Befunde sprechen für eine Unabhängigkeit der cyclischen Photophosphorylierung von der Atmungskette und für eine starke Übereinstimmung im Aufbau der Elektronentransportsysteme für die cyclische Photophosphorylierung bei R. rubrum und R. molischianum.

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Summary The isolated thylakoide-structures (chromatophores) of Rhodospirillum molischianum and Rhodospirillum rubrum are investigated with regard to activity of cyclic, light induced phosphorylation. A high activity of the photochemical apparatus needs an optimal external oxidation-reduction potential. Over-oxidation by oxygen or K₃[Fe[CN)₆] inhibit just as much as over-reduction by hydrogen and N-methyl phenazonium methosulfate or 2,6-dichlorphenol-indophenole both reduced with ascorbate. The highest activity is observed in hydrogen atmosphere without an electron-donator system or in helium with traces (0,07 mol) of succinate. The inhibitoryeffect of oxygen can partly be compensated by reduced PMS. The photochemical apparatus of R. molischianum and R. rubrum react nearly in the same way on changes of the external oxidation-reduction potential and both systems are strongly inhibited by antimycin A but not by low concentrations of 2,4-dinitrophenole. R. molischianum is a strict anaerobic organism and can grow only in the light and under low oxygen partialpressure. In comparison with it R. rubrum is more oxygen-tolerant. The strain can grow under conditions of aerobic dark metabolism. The present results together with those of other investigations provide strong evidence for the conclusion that the systems of cyclic photophosphorylation in both organisms have the same composition and are relatively independent from the respiratory chain.

     

Ferredoxin-dependent carbon assimilation in Rhodospirillum rubrum

Summary Evidence has been presented that a soluble fraction from R. rubrum cells contains two new primary carboxylation reactions which depend on the reducing power of ferredoxin: (a) pyruvate synthase which brings about a synthesis of pyruvate from acetyl-CoA and CO₂ and (b) -ketoglutarate synthase which brings about a synthesis of -ketoglutarate from succinyl-CoA and CO₂. The soluble fraction of R. rubrum cells contains also a series of other enzymes which, together with the ferredoxin-dependent enzymes, constitutes a reductive carboxylic acid cycle—a new cyclic pathway for CO₂ assimilation that was first found in the photosynthetic bacterium, Chlorobium thiosulfatophilum.Dedicated to C. B. van Niel on the occasion of his 70th birthday.Aided by grants from the National Institute of General Medical Sciences, Office of Naval Research and the National Science Foundation.     

Carbon monoxide dehydrogenase from Rhodospirillum rubrum

The carbon monoxide dehydrogenase from the photosynthetic bacterium Rhodospirillum rubrum was purified over 600-fold by DEAE-cellulose chromatography, heat treatment, hydroxylapatite chromatography, and preparative scale gel electrophoresis. In vitro, this enzyme catalyzed a two-electron oxidation of CO to form CO2 as the product. The reaction was dependent on the addition of an electron acceptor. The enzyme was oxygen labile, heat stable, and resistant to tryptic and chymotryptic digestion. Optimum in vitro activity occurred at pH 10.0. A sensitive, hemoglobin-based assay for measuring dissolved CO levels is presented. The in vitro Km for CO was determined to be 110 microM. CO, through an unknown mechanism, stimulated hydrogen evolution in whole cells, suggesting the presence of a reversible hydrogenase in R. rubrum which is CO insensitive in vivo.     

Ein neues Bacteriochlorophyll aus Rhodospirillum rubrum

Zusammenfassung Rhodospirillum rubrum und Rhodopseudomonas palustris enthalten ein Bacteriochlorophyll, das mit Farnesol statt mit Phytol veretert ist. Das neue Bacteriochlorophyll (Bchl a_F) läßt sich vom bekannten Bacteriochlorophyll a (Bchl a_P) durch ¹H-NMR-Spektroskopie unterscheiden sowie durch Dünnschichtchromatographie der entsprechenden Phäophytine an mit Silbernitrat imprägniertem Kieselgel. Die Chromophore von Bchl a_F und Bchl a_P sind auch bezüglich ihrer Stereochemie identisch.

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A new bacteriochlorophyll from Rhodospirillum rubrum

Summary Rhodospirillum rubrum and Rhodopseudomonas palustris contain a bacteriochlorophyll which is a farnesyl rather than a phytyl ester. The new bacteriochlorophyll (Bchl a_F) can be distinguished from the well known bacteriochlorophyll a (Bchl a_P) by ¹H-n.m.r. spectroscopy and by t.l.c. of the corresponding pheophytins on silver nitrate impregnated silica gel. The chromophores of Bchl a_P and Bchl a_F are identical in structure and stereochemistry.

     

D-alpha-Hydroxyglutarate dehydrogenase of Rhodospirillum rubrum.

D-alpha-Hydroxyglutarate dehydrogenase of R. rubrum grown anaerobically in the light was partially purified and some properties were investigated. 1. The enzyme catalyze stoichiometrically the dehydrogenation reaction of D-alpha-hydroxyglutarate into alpha-oxoglutarate, coupled with the reduction of 2, 6-dichlorophenolindophenol. 2. Cytochrome c2, cytochrome c, and ferricyanide are effective as electron acceptors with the crude enzyme but not with the purified one, whereas NAD+ and NADP+ are completely ineffective. The enzyme is thought to play a role in the electron transport system of the organism. 3. D-alpha-Hydroxyglutarate is virtually the sole substrate for the enzyme. The apparent activity against L-alpha-hydroxyglutarate is presumed to be due to contamination of the L-isomer sample with the D-isomer. The enzyme shows barely detectable activity against both isomers of malate and virtually no activity against DL-lactate and glycolate. 4. Both isomers of malate and oxalate, which are presumably substrate analogues, inhibit the enzyme activity. 5. The enzyme is not an inducible enzyme but rather is a constitutive one for R. rubrum, unlike from the enzyme of Pseudomonas putida which is an inducible enzyme for the catabolism of lysine.