13C NMR evidence for bacteriochlorophyll c formation by the C5 pathway in green sulfur bacterium, Prosthecochloris

The 13C NMR spectra of the pheophorbide of bacteriochlorophyll c, formed in the presence of L-[1-13C]glutamate and [2-13C]glycine and [13C]bicarbonate in Prosthecochloris aestaurii, were analysed. The isotope in the glutamate was specifically incorporated into the eight carbon atoms in the tetrapyrrole macrocycle derived from the C-5 of 5-aminolevulinic acid, while no specific enrichment of these eight carbon atoms was observed in the spectrum of the pigment formed in the presence of [2-13C]glycine. These labelling patterns provide evidence for the operation of the C5 pathway of 5-aminolevulinic acid synthesis for bacteriochlorophyll c formation in the bacterium. The labelling of bacteriochlorophyll c by [13C]bicarbonate is consistent with its formation from 5-[1,4,5-13C]aminolevulinic acid formed by the C5 pathway from [1,2,5-13C]glutamic acid. It is proposed that this glutamate is the transamination product of 2-[1,2,5-13C]oxoglutaric acid, arising by carboxylation of [1,4-13C]succinyl-CoA with 13CO2 catalysed by 2-oxoglutaric acid synthase activity, and that the labelled succinyl-CoA is, in turn, derived by the fixation of 13CO2 by the reductive tricarboxylic acid cycle. The 13C chemical shifts of two sp2 quaternary carbons of bacteriopheophorbide c methyl ester (C-2 and C-4) were reassigned.     

13C nuclear magnetic resonance studies on bacteriochlorophyll a biosynthesis in Rhodopseudomonas spheroides S

The 13C NMR spectra were analyzed in bacteriochlorophyll a and magnesium protoporphyrin methyl ester formed in Rhodopseudomonas spheroides S. in the presence of L-[1-13C]glutamate and [2-13C]glycine. After reassignment of three alpha-pyrrolic carbons (C-9, -14 and -16) of bacteriochlorophyll a, the spectra showed that C-2 of glycine was preferentially incorporated into the eight-carbon atoms in these tetrapyrrole macrocycles derived from C-5 of 5-aminolevulinic acid (ALA). C-2 of glycine was also incorporated specifically into methyl ester carbon of magnesium protoporphyrin IX methyl ester and methoxyl carbon of methoxycarbonyl group attached to isocyclic ring of bacteriochlorophyll a. No enrichment of these nine-carbon atoms was observed in the spectrum of bacteriochlorophyll formed in the presence of L-[1-13C]glutamate, showing exclusive operation of ALA synthase on bacteriochlorophyll biosynthesis.     

5-Aminolevulinic acid formation from glutamate via the C5 pathway in Clostridium thermoaceticum

A cell-free extract of the anaerobic eubacterium, Clostridium thermoaceticum, catalyzes the synthesis of 5-aminolevulinic acid (ALA) from glutamate via the C5 pathway. The enzyme reaction resembles that of higher plants and algae in cofactor requirements and sensitivity to ribonuclease. From the phylogenetic distribution it is proposed that the C5 pathway evolved earlier than the ALA synthase pathway.     

Ketogenesis in the living rat followed by 13C-NMR spectroscopy. Infusion of [1,3-13C]octanoate

13C-NMR spectroscopy was used as a noninvasive approach to study the metabolism of [1,3-13C]octanoate in rat liver. Using a properly adjusted surface coil a liver selection of better than 90% was achieved in the intact animal without abdominal surgery. After infusion of [1,3-13C]octanoate via the jugular vein different patterns of metabolites were observed depending on the physiological state of the rat. In the fasted animal, the major metabolites were those of the Krebs cycle while in the diabetic animal ketogenic end products were predominant. As a fatty acid of medium chain length octanoate is imported into the inner mitochondrial space without control by the carnitine acyl transferase system. Hence, the metabolic differences observed between diabetic and fasted rats result from an intramitochondrial control mechanism. The in vivo 13C-NMR results therefore support previous biochemical in vitro studies which concluded that a major control of ketone body production occurs in the inner mitochondrial space, presumably via the redox potential of the liver. As an unexpected result, 13C-NMR provides evidence for the transitory esterification of the infused 13C-labeled octanoic acid. The corresponding 13C-NMR chemical shifts are typical for glycerides.     

13C-NMR spectral study of reductively [13C]methylated glycophorin B

Glycophorin BN was reductively [13C]methylated and the 13C chemical shift of the N-terminal [13C]dimethyl-leucine residue was monitored as a function of pH. These results were compared to the pH-dependent chemical shift studies of the N-terminal [13C]dimethylleucine residues of intact glycophorin AN and N-terminal glyco-octapeptide AN. The results indicate that the titration data for [13C]dimethylleucine of glycophorin BN more closely resembles the titration data observed for the [13C]dimethylleucine residue of the N-terminal glyco-octapeptide AN rather than for the [13C]dimethylleucine residue of intact glycophorin AN. Integration of the 13C resonances indicated that glycophorin BN contains 3-4 lysine residues.     

Light environment and synthesis of bacteriochlorophyll by populations of Chromatium okenii under natural environmental conditions

Abstract: In the meromictic alpine Lake Cadagno a dense layer of phototrophic bacteria, mainly Chromatium okenii and Amoebobacter purpureus , develop annually at the chemocline at about 10 to 11 m depth. Radiometric spectral profiles of the incident sunlight demonstrate different attenuation coefficients in the mixolimnion and in the chemocline not only for the visible light effective at each depth (photosynthetically available radiation), but also for selected photosynthetically active wavelengths used by oxygenic and anoxygenic phototrophs. Phototrophic bacteria sampled from the upper part of the layer at the maximum of cell concentration were incubated in transparent bottles at the sampling depth and at a lower depth where the light intensity is only a few percent of the one at the sampling depth. Within 4 h the specific bacteriochlorophyll concentration (Bchl protein⁻¹) increased up to 50% depending on the difference in light intensity between the sampling and the incubation depth. The specific bacteriochlorophyll concentration in the upper part of the layer remained constant (53.0 mg Bchl g⁻¹ protein, S.D. = 4.8) in spite of large changes in cell concentrations in the lake water over the season. These observations illustrate the phenomenon of light-regulated pigment synthesis under natural conditions.     

Differences in the control of bacteriochlorophyll formation by light and oxygen

Control of bacteriochlorophyll formation was studied with continuous cultures of Rhodospirillum rubrum, Rhodopseudomonas sphaeroides, and Rhodopseudomonas capsulata. Oxygen controlled specific bacteriochlorophyll contents of the three species in a hyperbolical fashion irrespective of the presence of light. In Rps. sphaeroides, this applied to oxygen concentrations above 16% air saturation of the medium while at lower oxygen concentrations control followed a kinetics with negative cooperativity. Cell protein formation of R. rubrum and Rsp. sphaeroides was independent of oxygen concentrations while protein formation of Rps. capsulata increased at lower concentrations. Light controlled bacteriochlorphyll contents of R. rubrum and Rps. sphaeroides in a sigmoidal fashion. When growing at a constant low oxygen concentration cell protein formation increased with light energy flux in Rps. sphaeroides but remained unaffected in R. rubrum. Protein formation of R. rubrum increased with light energy flux only under anaerobic conditions. Two factor analyses were performed with R. rubrum and Rps. sphaeroides to study the combined effects of light and oxygen on bacteriochlorophyll formation. The results showed that both factors act independent of each other.Abbreviations ALA 5-aminolevulinic acid - R Rhodospirillum - Rsp. Rhodopseudomonas     

Synthesis and 13C-NMR analysis of 5 alpha- and 5 beta-cholestane-3,6-dione

Starting from cholesterol a simple and efficient synthesis of 5 alpha-cholestane-3,6-dione and 5 beta-cholestane-3,6-dione is described. The 13C shielding data of C-7, C-9, and C-19 in both isomers can be used in the determination of the stereochemistry at C-5 of these compounds. The combination of 13C NMR spectroscopy and the simple synthesis of both isomers offers good opportunities for the determination of the stereochemistry at C-5 of 3,6-dioxosteroids.     

Polyglucose synthesis in Chloroflexus aurantiacus studied by 13C-NMR

Chloroflexus aurantiacus OK-70 fl was grown photoautotrophically with hydrogen as electron source. The cultures were subjected to long term labelling experments with ¹³C-labelled acetate or alanine in the presence of sodium fluoroacetate. The presence of fluoroacetate caused the cells to accumulate large amounts of polyglucose which was hydrolysed and analysed by NMR. The labelling patterns of glucose were symmetric and in agreement with carbohydrate synthesis from acetate and CO₂ via pyruvate synthase. The content of carbon derived from added acetate was highest in C2 and C5 of glucose, at least 20% higher than in C1 and C6. About one third of the glucose carbon was derived from added acetate, the rest being from CO₂. Contrary to expectations, in glucose formed in the presence of C1-labelled acetate C1 and C6 contained more label than C2 and C5, and with C2-labelled acetate as the tracer glucose was mainly labelled in C2 and C5. Labelled CO₂ was formed from acetate labelled at either position. The labelling data indicate a new metabolic pathway in C. aurantiacus. It is suggested that the cells form C1-labelled acetyl-CoA from C2-labelled acetyl-CoA and vice versa by a cyclic mechanism involving concomitant CO₂ fixation and that this cycle is the part of the autotrophic CO₂ fixation pathways in C. aurantiacus in which acetyl-CoA is formed from CO₂.The polyglucose of C. aurantiacus appears to have predominantly (1–4)-linked structure with about 10% (1–6)-linkages as revealed by ¹³C-NMR.     

Binding of [1-13C]galactose-labeled N-acetyllactosamine to Erythrina cristagalli agglutinin as studied by 13C-NMR

The equilibrium binding kinetics of enzymatically prepared N-acetyllactosamine to the lectin from Erythrina cristagalli have been investigated by 13C-NMR spectroscopy. Under the experimental conditions used, NMR signals in the spectrum, corresponding to both the free and bound disaccharide species, were observed for the first time. This has permitted the simultaneous determinations of the equilibrium binding constant and the number of binding sites per lectin molecule. At the relatively high lectin concentrations used (0.3-0.87 mM), the association constants determined at 31 degrees C (approximately 6 X 10(3) M-1) are typically lower then those obtained by other methods employing much lower lectin concentrations. Extrapolation of the experimentally observed values to infinite dilution gave a better fit of the data (Ka approximately 1.4 X 10(4) M-1) with the binding constant determined by other methods (K approximately 1.1 X 10(4) M-1). The sugar residence time on the lectin (approximately 0.2 s) was determined directly from the signal's line-width using total line-shape analysis. Similar NMR experiments may permit an analysis of the interaction of the lectin with glycoproteins and cells labelled with 13C-enriched galactose residues. Moreover, information on lectin-galactose interactions at the binding site may be obtained by using galactose labeled at various carbons.     

Estimation of tissue phospholipids by natural abundance 13C-NMR

The total phospholipid content of excised rat muscle, liver, brain and kidney and of human muscle biopsies was estimated by natural abundance ¹³C-NMR after complete solubilization of the tissue membranes with excess halothane. An external dioxane capillary, calibrated against pure palmitic acid and phospholipid vesicles with known phosphate concentration, was inserted into the tissues, and the repeating methylene carbon peak area in the spectra of the halothane-treated tissues was integrated versus the dioxane reference peak area. The amount of tissue used for NMR analysis was quantitated by dry weight determination after ¹³C spectroscopy was completed. The phospholipid content estimated by the indirect NMR method was in good agreement with that measured by direct phosphate analysis and with literature data. For human muscle biopsies, the NMR method can also estimate the fraction of the total phospholipids which are mobile without treating the muscles with halothane. In this respect human muscles could be separated into three different groups: (1) normal and nonspecific muscle diseases, (2) myotonia and myopathy, (3) Duchenne dystrophy; with increasing fraction of the mobile phospholipids in this order.     

13C-NMR Spectra and Streochemistry of Isoechinulins A,B and C

¹³C-NMR spectra of isoechinulins A, B and C, metabolites from Aspergillus ruber, were fully assigned on the basis of chemical shifts and multiplicities and comparison with their analogues. Taking advantage of the symmetrical structure of the diketopiperazine ring, the stereochemistry of the trisubstituted carbon-carbon double bond in a dehydrotryptophyl moiety was determined as Z (cis) by measuring the coupling constants, , in the proton nondecoupled spectrum of isoechinulin B.     

Metabolic profiling by 13C-NMR spectroscopy: [1,2-13C2]glucose reveals a heterogeneous metabolism in human leukemia T cells

Metabolic profiling is defined as the simultaneous assessment of substrate fluxes within and among the different pathways of metabolite synthesis and energy production under various physiological conditions. The use of stable-isotope tracers and the analysis of the distribution of labeled carbons in various intermediates, by both mass spectrometry and NMR spectroscopy, allow the role of several metabolic processes in cell growth and death to be defined. In the present paper we describe the metabolic profiling of Jurkat cells by isotopomer analysis using (13)C-NMR spectroscopy and [1,2-(13)C(2)]glucose as the stable-isotope tracer. The isotopomer analysis of the lactate, alanine, glutamate, proline, serine, glycine, malate and ribose-5-phosphate moiety of nucleotides has allowed original integrated information regarding the pentose phosphate pathway, TCA cycle, and amino acid metabolism in proliferating human leukemia T cells to be obtained. In particular, the contribution of the glucose-6-phosphate dehydrogenase and transketolase activities to phosphoribosyl-pyrophosphate synthesis was evaluated directly by the determination of isotopomers of the [1'-(13)C], [4',5'-(13)C(2)]ribosyl moiety of nucleotides. Furthermore, the relative contribution of the glycolysis and pentose cycle to lactate production was estimated via analysis of lactate isotopomers. Interestingly, pyruvate carboxylase and pyruvate dehydrogenase flux ratios measured by glutamate isotopomers and the production of isotopomers of several metabolites showed that the metabolic processes described could not take place simultaneously in the same macrocompartments (cells). Results revealed a heterogeneous metabolism in an asynchronous cell population that may be interpreted on the basis of different metabolic phenotypes of subpopulations in relation to different cell cycle phases.     

13C-NMR of Clostridium pasteurianum ferredoxin after reductive methylation of the amines using [13C]formaldehyde

Clostridium pasteurianum 2(4Fe-4S) ferredoxin has been reductively methylated using [13C]formaldehyde and sodium cyanoborohydride. Lys3 and the N-terminal alanine, the only amines in the protein, are both dimethylated by this procedure. 13C-NMR titration of the apo, oxidized and reduced modified ferrodoxin indicate that the lysine pK is slightly over 10 in all three forms of the protein. In contrast, the N-terminal alanine shifts from a pK of 7.7 in the apoprotein to greater than 9 in both the oxidized and reduced modified ferredoxin. The unexpectedly high pK observed for the N-terminus is consistent with the presence of an ion pair in both the oxidized and reduced native forms of the protein. The methylated ferrodoxin is considerably less stable than the native protein, indicating an important role for the amines in protein stability.     

A 13C-methylation study of glycophorin A in intact erythrocytes by 13C-NMR spectroscopy

N-terminal $\text{N}{}^{\mathrm{\alpha }}\text{-[}{}^{13}\text{C]monomethylamino}$ derivatives for the N-terminal serine and leucine residues of glycophorins A^M and A^N, respectively, were obtained by reductively ¹³C-methylating homozygous human erythrocytes (MM, NN). The ¹³C-labeled glycophorins, A^M and A^N, were then isolated. A unique structural state was observed in solution reductively ¹³C-methylated glycophorin A^M that was not observed in glycophorin A^M derived from ¹³C-methylated erythrocytes. We attribute this state to the fact that some of the glycophorin A^M forms a head-to-head dimer when subjected to reductive ¹³C-methylation in aqueous solution. The ¹³C chemical shift data and pH titration data for the N-terminal [¹³C]dimethylamino and [¹³C]monomethylamino groups of glycophorin A^M and A^N derived from reductively ¹³C-methylated erythrocytes were in agreement with the chemical shift and titration data previously obtained for the N-terminal [¹³C]dimethylamino groups of solution reductively ¹³C-methylated glycophorins and related glycopeptides and peptides and N-terminal [¹³C]monomethylamino groups of related glycopeptides and peptides.     

In vivo 31P- and 13C-NMR studies of ATP synthesis and methane formation by Methanosarcina barkeri

Carbon and phosphorus metabolism of cell suspensions of Methanosarcina barkeri strain MS (DSM 800), grown on methanol, were probed in vivo by NMR. The experimental conditions, which involved thick cell suspensions, did not significantly affect the efficiency of the rate of methanol uptake by cells. Following exposure to methanol an acidification of both the intracellular and the extracellular spaces was observed and a gradient of 0.5 pH units across the cytoplasmic membrane was determined from the 31P-NMR data. High levels of intracellular ATP up to 4 mM were detected. The ADP concentration determined in a suspension of starved cells was only 2 mM, suggesting that a significant amount of ADP may be immobilized and is thus not detectable by NMR. In the presence of the protonophore, 3,3',4',5-tetrachlorosalicylanilide, the proton gradient was dissipated and the synthesis of ATP stopped. The inhibitor of the ATP synthase, N,N'-dicyclohexylcarbodiimide, was rather inefficient in inhibiting ATP synthesis. High concentrations of N,N'-dicyclohexylcarbodiimide (corresponding to 300 nmol/mg protein-1) were required to decrease the ATP content by approximately 60%, and, under these conditions, formation of acetyl phosphate was detected. However, the methanol consumption rate was not affected.     

Motional dynamics of residues in a beta-hairpin peptide measured by 13C-NMR relaxation.

Structurally characterizing partially folded peptides is problematic given the nature of their transient conformational states. 13C-NMR relaxation data can provide information on the geometry of bond rotations, motional restrictions, and correlated bond rotations of the backbone and side chains and, therefore, is one approach that is useful to assess the presence of folded structure within a conformational ensemble. A peptide 12mer, R1GITVNG7KTYGR12, has been shown to partially fold in a relatively stable beta-hairpin conformation centered at NG. Here, five residues, G2, V5, G7, Y10, G11, were selectively 13C-enriched, and 13C-NMR relaxation experiments were performed to obtain auto- and cross-correlation motional order parameters, correlation times, bond rotation angular variances, and bond rotational correlation coefficients. Our results indicate that, of the three glycines, G7 within the hairpin beta-turn displays the most correlated phi(t),psi(t) rotations with its axis of rotation bisecting the angle defined by the H-C-H bonds. These positively correlated bond rotations give rise to "twisting" type motions of the HCH group. V5 and Y10 phi,psi bond rotations are also positively correlated, with their CbetaCalphaH groups undergoing similar "twisting" type motions. Motions of near-terminal residues G2 and G11 are less restricted and less correlated and are best described as wobbling-in-a-cone. V5 and Y10 side-chain motions, aside from being highly restricted, were found to be correlated with phi,psi bond rotations. At 303 K, where the hairpin is considered "unfolded," the peptide exists in a transient, collapsed state because backbone and side-chain motions of V5, G7, and Y10 remain relatively restricted, unlike their counterparts in GXG-based tripeptides. These results provide unique information toward understanding conformational variability in the unfolded state of proteins, which is necessary to solve the protein folding problem.     

13C-NMR study of the ruthenium(II)-catalyzed degradation of labeled G-factor endoperoxides: proposal of a likely biogenetic pathway and evidence for CO2 release

G-factor endoperoxides are natural products acting as phytohormones and contribute to root inhibition and frost resistance in Eucalyptus grandis and other Myrtaceae. Several (Z)- and (E)-configured cyclopentane-1,3-dione metabolites of G-factors have been found in some Eucalyptus extracts. Interestingly, these probable metabolites are identical to the products obtained by Ru(II)-catalyzed reduction of G-factors. In the present work, we, thus, studied the mechanism involved in the formation of these compounds by means of in-depth NMR distribution analysis of two differently 13C-labeled G3-factors. The observed CO2 release is underlined, and a comparison with the corresponding Fe(II)-induced degradation of G-factor endoperoxides is made. Also, the type of electron transfer proposed in the two processes is discussed and, tentatively, connected to the role of endoperoxides in plants.