Carboxyl Group Of Acetate Research Articles

Novel quantitative method for individual isotopomer of organic acids from 13C tracer experiments determines carbon flow in acetogenesis

Anaerobic microbial acetogenesis is ubiquitous on Earth, and thus plays an important role in the global carbon cycle. The mechanism of carbon fixation in acetogens has attracted great interest from various studies for combatting climate change, and even for studying ancient metabolic pathways. Here, we developed a new, simple method for investigating carbon flows in the metabolic reaction of acetogen by conveniently and accurately determining the relative abundance of individual acetate- and/or formate-isotopomers formed in 13C labeling experiments. We measured the underivatized analyte by gas chromatography-mass spectrometry (GC-MS) coupled with a direct aqueous sample injection technique. The individual abundance of analyte isotopomers was calculated by the mass spectrum analysis using the least-squares approach. The validity of the method was demonstrated by determining known mixtures of unlabeled and 13C-labeled analytes. The developed method was applied to study the carbon fixation mechanism of the well-known acetogen Acetobacterium woodii grown on methanol and bicarbonate. We provided a quantitative reaction model for methanol metabolism of A. woodii, which indicated that methanol was not the sole carbon precursor of the acetate methyl group and that 20−22% of the methyl group was formed from CO2. In contrast, the carboxyl group of acetate appeared to form exclusively by CO2 fixation. Thus, our simple method, without laborious analytical procedures, has broad utility for the study of biochemical and chemical processes related to acetogenesis on Earth.

"Candidatus Galacturonibacter soehngenii" Shows Acetogenic Catabolism of Galacturonic Acid but Lacks a Canonical Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase Complex.

Acetogens have the ability to fixate carbon during fermentation by employing the Wood-Ljungdahl pathway (WLP), which is highly conserved across Bacteria and Archaea. In a previous study, product stoichometries in galacturonate-limited, anaerobic enrichment cultures of “Candidatus Galacturonibacter soehngenii,” from a novel genus within the Lachnospiraceae, suggested the simultaneous operation of a modified Entner-Doudoroff pathway for galacturonate fermentation and a WLP for acetogenesis. However, a draft metagenome-assembled genome (MAG) based on short reads did not reveal homologs of genes encoding a canonical WLP carbon-monoxide-dehydrogenase/acetyl-Coenzyme A synthase (CODH/ACS) complex. In this study, NaH13CO3 fed to chemostat-grown, galacturonate-limited enrichment cultures of “Ca. G. soehngenii” was shown to be incorporated into acetate. Preferential labeling of the carboxyl group of acetate was consistent with acetogenesis via a WLP in which the methyl group of acetate was predominately derived from formate. This interpretation was further supported by high transcript levels of a putative pyruvate-formate lyase gene and very low transcript levels of a candidate gene for formate dehydrogenase. Reassembly of the “Ca. G. soehngenii” MAG with support from long-read nanopore sequencing data produced a single-scaffold MAG, which confirmed the absence of canonical CODH/ACS-complex genes homologs. However, high CO-dehydrogenase activities were measured in cell extracts of “Ca. G. soehngenii” enrichment cultures, contradicting the absence of corresponding homologs in the MAG. Based on the highly conserved amino-acid motif associated with anaerobic Ni-CO dehydrogenase proteins, a novel candidate was identified which could be responsible for the observed activities. These results demonstrate operation of an acetogenic pathway, most probably as a yet unresolved variant of the Wood-Ljungdahl pathway, in anaerobic, galacturonate-limited cultures of “Ca. G. soehngenii.”

Probing the Origin of the Metabolic Precursor of the CO Ligand in the Catalytic Center of [NiFe] Hydrogenase

The O(2)-tolerant [NiFe] hydrogenases of Ralstonia eutropha are capable of H(2) conversion in the presence of ambient O(2). Oxygen represents not only a challenge for catalysis but also for the complex assembling process of the [NiFe] active site. Apart from nickel and iron, the catalytic center contains unusual diatomic ligands, namely two cyanides (CN(-)) and one carbon monoxide (CO), which are coordinated to the iron. One of the open questions of the maturation process concerns the origin and biosynthesis of the CO group. Isotope labeling in combination with infrared spectroscopy revealed that externally supplied gaseous (13)CO serves as precursor of the carbonyl group of the regulatory [NiFe] hydrogenase in R. eutropha. Corresponding (13)CO titration experiments showed that a concentration 130-fold higher than ambient CO (0.1 ppmv) caused a 50% labeling of the carbonyl ligand in the [NiFe] hydrogenase, leading to the conclusion that the carbonyl ligand originates from an intracellular metabolite. A novel setup allowed us to the study effects of CO depletion on maturation in vivo. Upon induction of CO depletion by addition of the CO scavenger PdCl(2), cells cultivated on H(2), CO(2), and O(2) showed severe growth retardation at low cell concentrations, which was on the basis of partially arrested hydrogenase maturation, leading to reduced hydrogenase activity. This suggests gaseous CO as a metabolic precursor under these conditions. The addition of PdCl(2) to cells cultivated heterotrophically on organic substrates had no effect on hydrogenase maturation. These results indicate at least two different pathways for biosynthesis of the CO ligand of [NiFe] hydrogenase.

Dinuclear and two- and three-dimensional gadolinium(III) complexes with mono- and dicarboxylate ligands: synthesis, structure and magnetic properties

Three new gadolinium(III) complexes with carboxylate ligands of formula [Gd2(ac)6(H2O)4]·2H2O (1), [Gd2(ac)2(fum)2(H2O)4]n (2) and [Gd2(ox)(fum)2(H2O)4]n·4nH2O (3) (ac = acetate, fum = fumarate and ox = oxalate) have been prepared and their structures determined by X-ray diffraction on single crystals. The structure of 1 is made up of discrete centrosymmetric di-µ-oxo(carboxylate acetate)digadolinium(III) units with an intramolecular Gd⋯Gd separation of 4.1589(3) A. Each gadolinium atom in 1 is nine-coordinated with two water molecules and seven carboxylate-oxygen atoms from four acetate ligands building a monocapped square antiprism environment. Compound 2 exhibits a sheet-like structure, the repeating intralayer motif being a rectangle of di-µ-oxo(carboxylate acetate)digadolinium(III) units [Gd⋯Gd separation of 3.8659(10) A] where the edges are defined by bridging bis-bidentate and tetrakis-monodentate fumarate groups. Each gadolinium atom in 2 is nine-coordinated with two water molecules and seven carboxylate oxygens from two acetate and three fumarate ligands building a distorted monocapped square antiprism. Compound 3 has a three-dimensional structure where chains of double oxo(carboxylate fumarate)-bridged gadolinium(III) ions are interlinked by bis-bidentate fumarate to afford layers which are further interconnected through bis-bidentate oxalate ligands. The corresponding values of the Gd⋯Gd separation are 4.5816(1), 8.2292(2) and 6.2989(1) A. Each gadolinium atom in 3 is ten-coordinated with two water molecules and eight oxygen atoms from four fumarate ligands and one oxalate group building a distorted bicapped square antiprism. Variable-temperature magnetic susceptibility measurements show the occurrence of weak but significant ferro- (1 and 3) and antiferromagnetic (2) interactions through the double oxo(carboxylate acetate) (1 and 2), double syn-syncarboxylate fumarate (2) and double oxo(carboxylate fumarate) and bis-bidentate oxalate (3) bridges. Monte Carlo calculations were performed in the case of 3 to simulate its magnetic data and to substantiate the best-fit values of the magnetic couplings through the different exchange pathways involved.

Structure of Activated Thrombin-Activatable Fibrinolysis Inhibitor, a Molecular Link between Coagulation and Fibrinolysis

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a metallocarboxypeptidase (MCP) that links blood coagulation and fibrinolysis. TAFI hampers fibrin-clot lysis and is a pharmacological target for the treatment of thrombotic conditions. TAFI is transformed through removal of its prodomain by thrombin-thrombomodulin into TAFIa, which is intrinsically unstable and has a short half-life in vivo. Here we show that purified bovine TAFI activated in the presence of a proteinaceous inhibitor renders a stable enzyme-inhibitor complex. Its crystal structure reveals that TAFIa conforms to the alpha/beta-hydrolase fold of MCPs and displays two unique flexible loops on the molecular surface, accounting for structural instability and susceptibility to proteolysis. In addition, point mutations reported to enhance protein stability in vivo are mainly located in the first loop and in another surface region, which is a potential heparin-binding site. The protein inhibitor contacts both the TAFIa active site and an exosite, thus contributing to high inhibitory efficiency.

Insights into crystallization mechanism: a synchrotron study of polymorphism in a cobalt acetate cluster compound

The structure of the title compound, di-mu3-acetato-1kappa2O,O':2kappaO';2kappaO:3kappa2O,O'-di-mu2-acetato-1kappaO:2kappaO';2kappaO:3kappaO'-octapyridyl-1kappa3N,2kappa2N,3kappa3N-tricobalt(II) bis(hexafluorophosphate), [Co3(C2H3O2)4(C5H5N)8](PF6)2, consists of divalent multinuclear cations in which three CoII ions are bridged by four mu2-acetate groups. The CoII ions are arranged in an approximately linear manner. The bridging acetates adopt two distinct coordination geometries: one pair bridges via a single O atom and the other pair employs both O atoms. The coordination octahedron around each CoII ion is completed by three pyridine molecules for the two outer CoII ions and by two for the inner ion. Charge is balanced by two PF6- anions. Single-crystal synchrotron X-ray studies indicate the existence of two polymorphs, both triclinic, which are distinguished primarily by differences in the relative orientations of the multinuclear cations, which in form 1 are tilted with respect to each other, but in form 2 are co-parallel as a result of the central Co atom lying on an inversion centre. The results of the structural studies allow an insight into the crystallization mechanism and resultant polymorphism. They suggest that a (bidentate carboxyl)C-O...H-C(pyridine) interaction exists in solution. For form 1, crystallized from pyridine, the interaction is not structure determining, as it is satisfied by interactions between solvent and solute. For form 2, crystallized from CH2Cl2, the interaction is between a bound acetate carboxyl group on one cation and a bound pyridine on another and is thus structure directing.

Pathways of methanol conversion in a thermophilic anaerobic (55 degrees C) sludge consortium.

The pathway of methanol conversion by a thermophilic anaerobic consortium was elucidated by recording the fate of carbon in the presence and absence of bicarbonate and specific inhibitors. Results indicated that about 50% of methanol was directly converted to methane by the methylotrophic methanogens and 50% via the intermediates H(2)/CO(2) and acetate. The deprivation of inorganic carbon species [Sigma(HCO(3)(-)+CO(2))] in a phosphate-buffered system reduced the rate of methanol conversion. This suggests that bicarbonate is required as an electron (H(2)) sink and as a co-substrate for the efficient and complete removal of the chemical oxygen demand. Nuclear magnetic resonance spectroscopy was used to investigate the route of methanol conversion to acetate in bicarbonate-sufficient and bicarbonate-depleted environments. The proportions of [1,2-(13)C]acetate, [1-(13)C]acetate and [2-(13)C]acetate were determined. Methanol was preferentially incorporated into the methyl group of acetate, whereas HCO(3)(-) was the preferred source of the carboxyl group. A small amount of the added H(13)CO(3)(-) was reduced to form the methyl group of acetate and a small amount of the added (13)CH(3)OH was oxidised and found in the carboxyl group of acetate when (13)CH(3)OH was converted. The recovery of [(13)C]carboxyl groups in acetate from (13)CH(3)OH was enhanced in bicarbonate-deprived medium. The small amount of label incorporated in the carboxyl group of acetate when (13)CH(3)OH was converted in the presence of bromoethanesulfonic acid indicates that methanol can be oxidised to CO(2 )prior to acetate formation. These results indicate that methanol is converted through a common pathway (acetyl-CoA), being on the one hand reduced to the methyl group of acetate and on the other hand oxidised to CO(2), with CO(2) being incorporated into the carboxyl group of acetate.

Structure and dynamics of metallomacrocycles: recognition of zinc xylyl-bicyclam by an HIV coreceptor.

As platforms for the design of metal-based therapeutic and diagnostic agents, macrocycles are rigid enough to provide strong metal binding sites and orient functional groups stereoselectively, yet flexible enough to accommodate structural changes required for induced-fit recognition of biological targets. We consider the recognition of the Zn(II) complex of the bis-tetraazamacrocycle xylyl-bicyclam, a potent anti-HIV agent, by the coreceptor CXCR4, a G-protein-coupled receptor used by HIV for membrane fusion and cell entry. NMR studies show that the macrocycles of Zn(II)(2)-xylyl-bicyclam perchlorate exist in aqueous solution as two major configurations, trans-I (nitrogen chirality R,S,R,S), and trans-III (S,S,R,R). Acetate addition induced a major structural change. X-ray crystallography shows that the acetate complex contains the unusual cis-V cyclam configuration (R,R,R,R and folded) with bidentate coordination of acetate to Zn(II) plus second-coordination-sphere double H-bond formation between diagonal NH protons on the opposite cyclam face and acetate carboxylate oxygens. Detailed 1D and 2D NMR studies show that the major configuration of Zn(II)(2)-xylyl-bicyclam acetate in aqueous solution is cis-V/trans-I. Molecular modeling shows that an analogous cis-V site can be formed when Zn(II)(2)-xylyl-bicyclam binds to CXCR4, involving the carboxylate groups of Asp262 (Zn(II) coordination) and Glu288 (double H-bonding). The second cyclam can adopt the trans-I (or trans-III) configuration with Zn(II) binding to Asp171. These interactions are consistent with the known structure-activity relationships for bicyclam anti-HIV activity and receptor mutation. Consideration of the anti-HIV activity of xylyl-bicyclam complexes of other metal ions suggests that affinity for carboxylates, configurational flexibility, and kinetic factors may all play roles in receptor recognition. For example, Pd(II) cyclam complexes interact only weakly with axial ligands and are inflexible and inactive, whereas Co(III) cyclams bind carboxylates strongly, are configurationally flexible, and yet have low activity. Our findings should aid the design of new generations of active macrocycles including highly specific chemokine receptor antagonists.

Metabolism of Dichloromethane by the Strict Anaerobe Dehalobacterium formicoaceticum.

The metabolism of dichloromethane by Dehalobacterium formicoaceticum in cell suspensions and crude cell extracts was investigated. The organism is a strictly anaerobic gram-positive bacterium that utilizes exclusively dichloromethane as a growth substrate and ferments this compound to formate and acetate in a molar ratio of 2:1. When [C]dichloromethane was degraded by cell suspensions, formate, the methyl group of acetate, and minor amounts of methanol were labeled, but there was no nuclear magnetic resonance signal corresponding to the carboxyl group of acetate. This finding and previously established carbon and electron balances suggested that dichloromethane was converted to methylene tetrahydrofolate, of which two-thirds was oxidized to formate while one-third gave rise to acetate by incorporation of CO(2) from the medium in the acetyl coenzyme A synthase reaction. When crude desalted extracts were incubated in the presence of dichloromethane, tetrahydrofolate, ATP, methyl viologen, and molecular hydrogen, dichloromethane and tetrahydrofolate were consumed, with the concomitant formation of stoichiometric amounts of methylene tetrahydrofolate. The in vitro transfer of the methylene group of dichloromethane onto tetrahydrofolate required substoichiometric amounts of ATP. The reaction was inhibited in a light-reversible fashion by 20 muM propyl iodide, thus suggesting involvement of a Co(I) corrinoid in the anoxic dehalogenation of dichloromethane. D. formicoaceticum exhibited normal growth with 0.8 mM sodium in the medium, and crude extracts contained ATPase activity that was partially inhibited by N,N'-dicyclohexylcarbodiimide and azide. During growth with dichloromethane, the organism thus may conserve energy not only by substrate-level phosphorylation but also by a chemiosmotic mechanism involving a sodium-independent F(0)F(1)-type ATP synthase.

Solution (sup13)C Nuclear Magnetic Resonance Spectroscopic Analysis of the Amino Acids of Methanosphaera stadtmanae: Biosynthesis and Origin of One-Carbon Units from Acetate and Carbon Dioxide

We found that general pathways for amino acid synthesis of Methanosphaera stadtmanae, a methanogen that forms CH(inf4) from H(inf2) and methanol, resembled those of methanogens that form CH(inf4) from CO(inf2) or from the methyl group of acetate. We determined the incorporation of (sup14)C-labeled CO(inf2), formate, methanol, methionine, serine, and acetate into cell macromolecules. Labeling of amino acid carbons was determined by solution nuclear magnetic resonance spectroscopy after growth with (sup13)C-labeled acetate, CO(inf2), serine, and methanol. The (alpha) and (beta) carbons of serine and alanine were formed from carboxyl and methyl carbons of acetate, respectively, and the amino acid carboxyl groups were formed from CO(inf2). This indicates that pyruvate was formed by reductive carboxylation of acetate. Labeling of the methyl carbon of methionine indicated that the major route of synthesis was from the hydroxymethyl carbon of serine that arises from the methyl carbon of acetate. Methanol was a minor source of the methyl of methionine. Unambiguous assignment was made of the sources of all carbons of histidine. Labeling of the histidine 7 position ((epsilon) carbon) was consistent with formation from the C-2 of the purine ring of ATP and the origin of the C-2 from a formyl unit derived from the hydroxymethyl carbon of serine.

Mesophilic syntrophic acetate oxidation during methane formation by a triculture at high ammonium concentration

In a mesophilic (37°C) triculture at a high ammonium concentration and pH8, methanogenesis from acetate occurred via syntrophic acetate oxidation. Studies with 14C-labelled substrates showed that the amount of labelled methane formed from 1-14C-labelled acetate was equal to that formed from 2-14C-labelled acetate. Labelled methane was also formed from H14CO3-. These results clearly showed that both the methyl and carboxyl groups of acetate were oxidized to CO2 and that CO2 was reduced to CH4 through hydrogenotrophic methanogenesis. During growth of the triculture, a significant isotopic exchange between the carboxyl group of acetate and bicarbonate occurred. As a result, there was a decrease in the specific activity of 1-14C-acetate, and the production of 14CO2 was slightly higher from 1-14C- than from 2-14C-acetate. For each mole acetate degraded, 0.94 mol methane was formed; 9.2 mmol acetate was metabolized during the 294 days of incubation.

Carbon isotopic composition of bacterial methane in a soil incubation experiment: Contributions of acetate and [formula omitted

Anaerobic incubations of paddy soil collected from Konosu, Japan, were carried out for 10 weeks to clarify the general principles that govern the variation of carbon isotopic composition of bacterial methane from freshwater areas. The concentrations and isotopic compositions of CH 4, CO 2, and acetate produced in the incubated system were measured. The δ 13 C value of biogenic CH 4 was highly variable ranging from −60 to −33%., corresponding to changes in its formation pathways: acetate fermentation and CO 2 H 2 reduction. Acetate accumulated only during the first week. After it became depleted, acetate production completely limited CH 4 production. The δ 13 C value of the methyl carbon of acetate, which was accumulated with addition of inhibitor for methanogenesis, ranged from −43 to −30%.. The δ 13 C value of methane from acetate was estimated to be −43 to −30%., after acetate depletion. The δ 13 C value of CH 4 from CO 2 H 2 reduction was estimated to be −77 to −60%.. An enrichment in 13C of CO 2 localized around the site of methanogenesis was suggested. CO 2 produced in situ during methanogenesis (e.g., CO 2 from carboxyl group of acetate) was possibly utilized as well. Using the above δ 13 C values of two endmembers of CH 4, the change of acetate contribution for CH 4 production was calculated: less than 12% until week 1, 65 to 100% at weeks 1 to 3, 16 to 28% at weeks 3 to 5, and 35 to 40% after week 5. Variability of CH 4 δ 13 C resulted from the difference in contribution of each biological process. Also, the δ 13 C value of CH 4 was a useful indicator for assessing the contribution of each process to methane production in sulfate depleted freshwater areas.

CO2reduction to acetate in anaerobic bacteria

The reduction of 2 CO2 to acetate is catalyzed in the energy metabolism of homoacetogenic bacteria, which couple acetate formation to the synthesis of ATP. The carboxyl group of acetate is formed from CO2 via reduction to a bound carbonyl ([CO]), a redution that requires the input of methaolic energy when hydrogen is used as the electron donor. The methyl group of acetate is formed via formate and tetrahydrofolate bound C1 intermediates including methyl tetrahydrofolate as the intermediates. The methyl group is the ‘condensed’ with the carbonyl and CoA to acetyl-CoA, which is converted to acetate in the energy metabolism or to cell carbon in the anabolism of the bacteria. The mechanism of ATP synthesis coupled to CO2 reduction to acetate is still unclear. The only reaction sufficiently exergonic is the reduction of methylene tetrahydrofolate to methyl tetrahydrofolate. Indirect evidence was presented that this reaction in homoacetogens might be coupled to the electrogenic transport of sodium across the cytoplasmic membrane. The sodium gradient formed via methylene-THF reduction could be transformed into a proton gradient via a sodium/proton antiporter. ATP would then be synthesized by a proton translocating ATP synthase.

CO 2 reduction to acetate in anaerobic bacteria

The reduction of 2 CO 2 to acetate is catalyzed in the energy metabolism of homoacetogenic bacteria, which couple acetate formation to the synthesis of ATP. The carboxyl group of acetate is formed from CO 2 via reduction to a bound carbonyl ([CO]), a redution that requires the input of methaolic energy when hydrogen is used as the electron donor. The methyl group of acetate is formed via formate and tetrahydrofolate bound C 1 intermediates including methyl tetrahydrofolate as the intermediates. The methyl group is the ‘condensed’ with the carbonyl and CoA to acetyl-CoA, which is converted to acetate in the energy metabolism or to cell carbon in the anabolism of the bacteria. The mechanism of ATP synthesis coupled to CO 2 reduction to acetate is still unclear. The only reaction sufficiently exergonic is the reduction of methylene tetrahydrofolate to methyl tetrahydrofolate. Indirect evidence was presented that this reaction in homoacetogens might be coupled to the electrogenic transport of sodium across the cytoplasmic membrane. The sodium gradient formed via methylene-THF reduction could be transformed into a proton gradient via a sodium/proton antiporter. ATP would then be synthesized by a proton translocating ATP synthase.

Metabolism of One-Carbon Compounds by the Ruminal Acetogen Syntrophococcus sucromutans

Syntrophococcus sucromutans is the predominant species capable of O demethylation of methoxylated lignin monoaromatic derivatives in the rumen. The enzymatic characterization of this acetogen indicated that it uses the acetyl coenzyme A (Wood) pathway. Cell extracts possess all the enzymes of the tetrahydrofolate pathway, as well as carbon monoxide dehydrogenase, at levels similar to those of other acetogens using this pathway. However, formate dehydrogenase could not be detected in cell extracts, whether formate or a methoxyaromatic was used as electron acceptor for growth of the cells on cellobiose. Labeled bicarbonate, formate, [1-C] pyruvate, and chemically synthesized O-[methyl-C]vanillate were used to further investigate the catabolism of one-carbon (C(1)) compounds by using washed-cell preparations. The results were consistent with little or no contribution of formate dehydrogenase and pointed out some unique features. Conversion of formate to CO(2) was detected, but labeled formate predominantly labeled the methyl group of acetate. Labeled CO(2) readily exchanged with the carboxyl group of pyruvate but not with formate, and both labeled CO(2) and pyruvate predominantly labeled the carboxyl group of acetate. No CO(2) was formed from O demethylation of vanillate, and the acetate produced was position labeled in the methyl group. The fermentation pattern and specific activities of products indicated a complete synthesis of acetate from pyruvate and the methoxyl group of vanillate.

Anaerobic acetate oxidation to CO2 by Desulfotomaculum acetoxidans

Desulfotomaculum acetoxidans oxidizes acetate to CO2 with sulfate. This organism metabolizes acetate via a pathway in which C1 units rather than tri- and dicarboxylic acids are intermediates. We report here that cell extracts of D. acetoxidans catalyzed an exchange between CO2 and the carboxyl group of acetate at a rate of 90 nmol · min-1 · mg-1 protein which is sufficient to account for the in vivo acetate oxidation rate of 250 nmol · min-1 · mg-1 protein. The reaction was strictly dependent on both ATP and coenzyme A. The extracts contain high activities of acetate kinase (6.3 U · mg-1 protein) and phosphotransacetylase (60 U · mg-1 protein). These findings indicate that acetyl-CoA rather than acetyl-phosphate or acetate is the substrate of the carbon-carbon cleavage activity. Exchange was only observed in the presence of strong reducing agents such as Ti3+. Interestingly, the cell extracts also catalyzed the reduction of CO2 to CO with Ti3+ as electron donor (120 nmol · min-1 · mg-1 protein). Carbon monoxide dehydrogenase and other oxidoreductases involved in acetate oxidation were found to be partially associated with the membrane fraction suggesting a membrane localization of these enzymes.

Calcium complexation with a highly calcium selective chelator: crystal structure of ca(CaFBAPTA) ·5H 2O

The structure of the complex of calcium ions with 5,5'-difluoro-1,2- bis( o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (FBAPTA) has been determined. The calcium complex, with stoichiometry Ca(CaFBAPTA) · 5H 2O crystallizes in a monoclinic space group P2 1/ c with 4 formula units/unit cell with a(Å) = 9.767, b(Å) = 26.309, c(Å) = 11.769; β (deg) = 111.57. The structure may be considered as the calcium salt of a CaFBAPTA2- complex in which the octacoordinate calcium ion is complexed with the two ether oxygen atoms, the two nitrogens, and coordinated in a unidentate manner with the four acetate carboxyl groups. The geometry of the nitrogen atoms is nearly tetrahedral and provides a basis for the interpretation of the optical perturbations which result from calcium complexation with BAPTA type ligands. In addition, it is clear from the distance and relative orientation of the aromatic rings that ring current contributions to the fluorine chemical shifts are of negligible importance, so that such effects cannot be used in the design of NMR active indictators. Although BAPTA and analogs such as PBAPTA and quin2 have found extensive physiological applications as a consequence of the high degree of selectivity for calcium ions over magnesium ions which they exhibit, this is the first reported structural data for any of these complexes.

Carbon monoxide metabolism in cell suspensions of Peptostreptococcus productus strain Marburg

Abstract Peptostreptococcus productus strain Marburg is able to grow on CO as the sole energy source and converts this substrate to acetate and CO 2 according to the following equation: 4 CO + 2 H 2 O → 1 acetate − + 1 H + + 2 CO 2 In cell suspensions of the organism the apparent K m value for CO conversion to acetate was about 270 μM (∼ 25% in the gas phase at 140 kPa). Cells incubated in the presence of 14 CO and unlabelled CO 2 incorporated CO specifically into the carboxyl group of acetate like other acetogenic bacteria tested. However, in contrast to experiments performed with other acetogens, 100% of the carboxyl group formed was derived from CO rather than from CO 2 indicating that ‘free’ CO rather than a bound carbonyl formed from CO 2 may be the precursor for the synthesis of the carboxyl group of acetate in this organism.

Anaerobic catabolism of formate to acetate and CO2 by Butyribacterium methylotrophicum.

The catabolism of sodium formate to acetate and carbon dioxide by the anaerobic acetogen Butyribacterium methylotrophicum was analyzed by fermentation time course and 13C nuclear magnetic resonance studies. Significant hydrogen production and consumption fluxes were observed during formate catabolism but not during the catabolism of formate plus CO. In the latter case, formate and CO were simultaneously consumed and label distribution studies with mixtures of 13C-labeled CO and formate demonstrated their preferential incorporation into the acetate carboxyl and methyl groups, respectively. Hydrogen consumption was inhibited by CO when both were present, whereas hydrogen and formate were simultaneously consumed when CO2 was supplied. Carbon dioxide was required for the conversion of CO to acetate, but a similar need was not observed when methanol plus CO or formate plus CO was present. These analyses indicate a bifurcated single-carbon catabolic pathway in which CO2 is the sole single-carbon compound that directly supplies the carbonyl and methyl group synthesis pathways leading to the formation of acetyl coenzyme A, the primary reduced product. We discuss causes for the reported inability of B. methylotrophicum to use formate as a sole substrate.

Methanogenesis from Acetate by Methanosarcina barkeri: Catalysis of Acetate Formation from Methyl Iodide, CO2 , and H2 by the Enzyme System Involved

Cell suspensions of Methanosarcina barkeri grown on acetate catalyze the formation of methane and CO2 from acetate as well as an isotopic exchange between the carboxyl group of acetate and CO2. Here we report that these cells also mediate the synthesis of acetate from methyl iodide, CO2, and reducing equivalents (H2 or CO), the methyl group of acetate being derived from methyl iodide and the carboxyl group from CO2. Methyl chloride and methyltosylate but not methanol can substitute for methyl iodide in this reaction. Acetate formation from methyl iodide, CO2, and reducing equivalents is coupled with the phosphorylation of ADP. Evidence is pres­ented that methyl iodide is incorporated into the methyl group of acetate via a methyl corrinoid intermediate (deduced from inhibition experiments with propyl iodide) and that CO2 is assimi­lated into the carboxyl group via a C1 intermediate which does not exchange with free formate or free CO. The effects of protonophores, of the proton-translocating ATPase inhibitor N.N′-di- cyclohexylcarbodiimide, and of arsenate on acetate formation are interpreted to indicate that the reduction of CO2 to the oxidation level of the carboxyl group of acetate requires the presence of an electrochemical proton potential and that acetyl-CoA or acetyl-phosphate rather than free acetate is the immediate product of the condensation reaction. These results are discussed with respect to the mechanism of methanogenesis from acetate.