RESEARCH
Bacterium fixes C02 by reversing Krebs cycle A new kind of photosynthesis uses ferredoxin, a nonporphyrin, to reduce carbon dioxide to carboxylic acids A new cyclic pathway for fixing carbon dioxide has been found in a photosynthetic bacterium at the University of California, Berkeley, department of cell physiology. Dr. M. C. W. Evans, Dr. B. B. Buchanan, and Dr. D. I. Anion have found evidence for a re ductive carboxylic acid cycle in Chlorobiam thiosulfatophilum. The inter mediates in the cycle have the carbon skeletons of a variety of amino acids. Part of this cyclic pathway reverses the Krebs citric acid cycle. The Krebs cycle is an important degradative mechanism for making amino acid precursors in nonphotosynthetic cells. Unlike the Krebs cycle, which re leases energy, the reductive carboxylic acid cycle requires energy. This en ergy is supplied by reduced ferredoxin and adenosine triphosphate (ATP). Dr. Anion will describe the new cycle this week at the National Academy of Sciences meeting, in Washington, D.C. Until now, the only cyclic pathway known for carbon fixation in photosynthetic cells was the reductive pentose phosphate cycle. Berkeley's Dr. Melvin Calvin and co-workers discovered the pentose cycle more than a decade ago. In the Calvin cycle, which still seems to be the main route for carbon assimilation by plants, carbon dioxide combines with ribulose diphosphate. The resulting 3-phosphoglyceric acid (PGA) gains another phosphate group from ATP and is then reduced to triose phosphate by reduced triphosphopyridine nucleotide (TPNH 2 ). To complete the cycle, ribulose diphos phate is regenerated through another series of enzyme-catalyzed reactions. It recycles by incorporating another molecule of carbon dioxide. Over-all, one molecule of carbon dioxide becomes the equivalent of one third of a molecule of triose phos phate. The cell can convert triose phosphate to glucose and thence to starch. By contrast, the new cycle converts four molecules of carbon di oxide into one molecule of a C 4 dicarboxylic acid. While carbon assimilation in many plants leads mostly to carbohydrates, photosynthetic bacteria produce mainly amino acids. Although the re ductive pentose phosphate cycle pro 50 C&EN APRIL 25, 1966
vides a path from PGA to amino acids, the new reductive carboxylic acid cy cle is a more direct pathway to amino acids. Its intermediates have the car bon skeletons of several key amino acids. By a single animation, for example, α-ketoglutarate from the cycle could go to glutamate; pyruvate could go to alanine; and oxalacetate could go to aspartate. Heme compounds can be made from succinyl coenzyme A, another intermediate in the cycle. Fats can be made from acetyl coen zymes A. The Berkeley group has found evidence for the cycle only in C. thiosulfatophilum, but they think it probably operates in other photosynthetic bacteria as well. Other workers have reported that the Calvin cycle also operates in C. thiosulfatophilum. The Calvin cycle may be making sugars in photosyn thetic bacteria while the reductive carboxylic acid cycle produces amino acids and precursors of lipids and por phyrins. However, the new cycle also yields phosphoenolpyruvate, which could readily form carbohydrates. The new cycle depends on the re ducing power of ferredoxins, which constitute a class of low-molecularweight iron-proteins whose structures aren't fully known. Unlike the cyto chromes (a group of iron-proteins that function in cellular catabolism) ferre doxins don't contain a porphyrin
group. On present evidence, ferre doxin doesn't appear to enter directly into carbon fixation in plants. It regenerates TPNHo and catalyzes the formation of ATP. Ferredoxin, though, is a far more powerful reducing agent than TPNHo. It is the most powerful reducing agent known in cellular chemistry. In 1964, Dr. R. Bachofen, Dr. Buchanan, and Dr. Anion discovered that, in the bac teria Chromatium and Clostridium pasteurianum, ferredoxin acts directly in a carbon fixation reaction—the carboxylation of acetyl coenzyme A to pyruvate (C&EN, Sept. 21,1964, page 48). They also identified in these bac teria a previously unknown enzyme, pyruvate synthase, which catalyzes the carboxylation.
Another ferredoxin carboxylation. Last year, Dr. Buchanan and Dr. Evans found another ferredoxin-dependent carbon fixation in Chlorobium—the carboxylation of succinyl coenzyme A to α-ketoglutarate. Here again, ferredoxin and a newly found enzyme, α-ketoglutarate synthase, re verse what had been thought to be an irreversible decarboxylation in the Krebs cycle. Having established these two key carboxylations, the California group searched for a complete cycle which would incorporate four molecules of carbon dioxide. The other two car boxylations—phosphoenolpyruvate to
Potent ferredoxin reverses decarboxylations of a-keto acids
NMR gives precise cyclobutane structure Near-planar molecule bends at 18°; methylenes tilt at 6°
TRACERS. Dr. Β. Β. Buchanan, Dr. D. I. Arnon, and Dr. M. C. W. Evans (left to right) at Berkeley traced the new photosynthetic path by feeding radio active COo to bacteria in these jugs
oxalacetate, and α-ketoglutarate to isocitrate—pose no thermodynamic prob lems and don't depend directly on ferredoxin. To learn whether this pathway is followed in a photosynthetic bacte rium, the Berkeley workers first con firmed that C. thiosulfatophilum con tains the 17 enzymes required by the cycle. They incubated extracts of the bacterium with carbon-14 labeled substrates and identified the products by paper chromatography and radioautography. The activities of these enzymes, measured in the direction of the cycle, seem adequate to drive it in the proper direction. The lowest rate of activity found for any enzyme in the extracts was about one tenth of the rate of carbon dioxide fixation by whole cells. Other workers have re ported that the lowest rate of activity in extracts of enzymes in the Calvin cycle is about one thirtieth of the rate of fixation in whole cells. The California workers traced the new pathway by exposing suspensions of the bacterium to labeled carbon di oxide and identifying the products of the labeled carbon at various time in tervals. Glutamate was the first stable product they identified. It accounted for three quarters of the labeled car bon fixed in the first 30 seconds. (Glutamate is one of the main prod ucts of photosynthetic bacteria. It can be converted to other amino acids in the cell.) Small amounts of suc cinate and aspartate also appear quickly and, like glutamate, gradually decline. No more than 10% of the labeled carbon fixed went into phos phate esters. All these results are consistent with the new cycle.
A precise determination of the molec ular structure of cyclobutane has been made from nuclear magnetic reso nance spectroscopy in a nematic sol vent by two scientists at Bell Tele phone Laboratories, Murray Hill, N.J. Dr. Saul Meiboom and Dr. Lawrence C. Snyder have found that by measur ing the NMR spectrum of cyclobutane dissolved in p,p'-di-n-hexyloxyazoxybenzene, they can determine cyclobutane's dihedral angle and the angle of tilt of the methylene group. Some organic substances exist in one or more liquid crystalline phases at temperatures between those where the solid phase and the normal isotropic liquid phase occur. The three main types of liquid crystals are nematic, smectic, and cholesteric. In the ne matic phase, molecules tend to orient themselves under the influence of a magnetic field, as in an NMR spec trometer. In such cases, the axis of highest susceptibility of the molecular aggregates is parallel to the field. Dr. Alfred Saupe of Physikalisches Institut der Universitàt, Freiburg, West Germany, and Dr. Gerhard Englert of Hoffmann-La Roche & Co., Ltd., Basel, Switzerland, earlier found that molecules in a nematic solvent experience an anisotropic environment and give highly resolved NMR spectra (C&EN, March 16, 1964, page 42). The NMR lines are relatively sharp because the dissolved molecules are sufficiently mobile to average dipoledipole interactions with the solvent. The anisotropy of the molecular motion leads to large intramolecular mag-
netic dipole-dipole interactions between nuclei, and to a corresponding fine structure in the NMR spectrum. The most important terms in the spin Hamiltonian that Dr. Meiboom and Dr. Snyder used in their molecular structure determinations in nematic solvents are the direct spin-spin couplings. To determine the structure of a molecule dissolved in a nematic solvent, the Bell scientists first assume a certain geometry and motion for the molecule. Using the spin Hamiltonian, they compute a theoretical NMR spectrum and compare it with the experimental NMR spectrum of the molecule in the nematic medium. If the experimental and calculated spectra do not agree, the assumed geometry and motion for the molecule are incorrect. A new spectrum is then calculated for a different geometry and motion. This procedure is repeated until close agreement between calculated and experimental NMR spectra are obtained. This method can be used to determine molecular structure with all distances determined relative to one other distance. Dr. Meiboom and Dr. Snyder tested their method on cyclopropane, a molecule whose structure has been determined by electron diffraction. They used ρ,ρ'-di-n-hexyloxyazoxybenzene as the nematic solvent. With the as sumption that the distance between a pair of protons on the same side of the cyclopropane ring has the electron dif fraction value of 2.521 Α., they ob tained values of 1.485 A. for the C—C distances, 1.106 A. for the C-H dis-
OTHERS. Using NMR spectra obtained in nematic solvents, Dr. Saul Mei boom (seated) and Dr. L. C. Snyder are now studying the structure of simple organic molecules in addition to cyclopropane and cyclobutane APRIL 25, 1966 C&EN
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