ABSTRACT
The effects of metformin on invertase activity and its inhibition on sucrose digestion were studied. The rapid unfolding kinetics of invertases, followed a two-state model with an inactive intermediate formation. The dynamic interaction between metformin and invertase caused the secondary structure of the enzyme to become less ß-sheet, more α-helix, and random coiling oriented, which weakened the binding force between enzyme and its substrate. Metformin acted as a chaotrope and disrupted the hydrogen bonds of water, which facilitated the unfolding of invertase. However, some sugar alcohols, which promoted the H-bond formation of water, could repair the secondary structure of metformin-denatured invertase and therefore regulate the enzyme activity. This research enriches our understanding of the mechanism of enzyme unfolding induced by guanidine compounds. Moreover, because metformin and sugar substitutes are of concern to diabetes, this research also provides useful information for understanding the activity of the digestive enzyme that coexists with metformin and sugar alcohols.
Subject(s)
Metformin , beta-Fructofuranosidase , Metformin/chemistry , Metformin/pharmacology , Kinetics , beta-Fructofuranosidase/chemistry , beta-Fructofuranosidase/metabolism , Sucrose/chemistry , Sucrose/metabolism , Protein Unfolding/drug effects , Hydrogen Bonding , Protein Structure, Secondary , Digestion/drug effectsABSTRACT
Stopped-flow fluorescence spectroscopy is a highly sensitive method for measuring rapid enzyme kinetics. A wide range of fluorophores can be employed, and fluorescence and fluorescence polarization can be measured. Thus, binding, conformational changes, and catalysis can, in principle, be measured, making it helpful in probing the entire kinetic landscape of a reaction. In this chapter, we use the bacterial RNA processing enzyme ribonuclease P (RNase P) as a model system to illustrate the determination of the kinetic constants for substrate binding and cleavage, thus allowing mechanistic questions regarding the effects of reaction conditions, mutations, or drug binding to be answered.
Subject(s)
Fluorescence Polarization , Ribonuclease P , Spectrometry, Fluorescence , Kinetics , Fluorescence Polarization/methods , Ribonuclease P/metabolism , Ribonuclease P/chemistry , Spectrometry, Fluorescence/methodsABSTRACT
The practical application of aqueous zinc-ion batteries for large-grid scale systems is still hindered by uncontrolled zinc dendrite and side reactions. Regulating the electrical double layer via the electrode/electrolyte interface layer is an effective strategy to improve the stability of Zn anodes. Herein, we report an ultrathin zincophilic ZnS layer as a model regulator. At a given cycling current, the cell with Zn@ZnS electrode displays a lower potential drop over the Helmholtz layer (stern layer) and a suppressed diffuse layer, indicating the regulated charge distribution and decreased electric double layer repulsion force. Boosted zinc adsorption sites are also expected as proved by the enhanced electric double-layer capacitance. Consequently, the symmetric cell with the ZnS protection layer can stably cycle for around 3,000 h at 1 mA cm-2 with a lower overpotential of 25 mV. When coupled with an I2/AC cathode, the cell demonstrates a high rate performance of 160 mAh g-1 at 0.1 A g-1 and long cycling stability of over 10,000 cycles at 10 A g-1. The Zn||MnO2 also sustains both high capacity and long cycling stability of 130 mAh g-1 after 1,200 cycles at 0.5 A g-1.
ABSTRACT
The subjective measurement of the dynamic perception of sweetness is a problem in food science. Herein, the rapid interactions of sugars and sugar alcohols with sweet taste receptors on living cells on a millisecond timescale were studied via stopped-flow fluorescence spectroscopy. According to the rapid-kinetic parameters, sweeteners were divided into two groups. Sweeteners in group I disrupted the hydrogen bond network structure of water, and the apparent rate constant (kobs) was in the range of 0.45-0.6 s-1. Sweeteners in group II promoted the hydrogen bond formation of water, and the kobs was mostly in the range of 0.6-0.75 s-1. For most sweeteners, the kobs of cell responses was negatively correlated with the apparent specific volume of sweeteners. The differences in the cellular responses may be attributed to the disturbance in the water structure. Experimental results showed that the kinetic parameters of sweet cell responses reflected the dynamic perception of sweetness. Rapid kinetics, solution thermodynamic analysis, and water structure analysis enriched the physicochemical study of the sweetness mechanism and can be used to objectively evaluate the dynamic perception of sweetness.
ABSTRACT
Although MoO2-based electrodes have been intensively studied as potential candidate anodes for lithium-ion batteries (LIBs) based on their high theoretical capacity (840 mAh g-1 and 5447 mAh cm-3), common issues such as severe volume variation, electrical conductivity loss, and low ionic conductivity, are prevalent. In this study, we demonstrate enhanced Li-ion kinetics and electrical conductivity of MoO2-based anodes with ternary MoO2-Cu-C composite materials. The MoO2-Cu-C was synthesized via two-step high energy ball milling where Mo and CuO are milled, followed by the secondary milling with C. With the introduction of the Cu-C hybrid matrix in MoO2 nanoparticles via the element transfer method using mechanochemical reactions, the sluggish Li-ion diffusion and unstable cycling behavior were significantly improved. The inactive Cu-C matrix contributes to the increase in electrical and ionic conductivity and mechanical stability of active MoO2 during cycling, as characterized by various electrochemical analyses and ex situ analysis techniques. Hence, the MoO2-Cu-C anode delivered promising cycling performance (674 mAh g-1 (at 0.1 A g-1) and 520 mAh g-1 (at 0.5 A g-1), respectively, after 100 cycles) and high-rate property (73% retention at 5 A g-1 as comparison with the specific capacity at 0.1 A g-1). The MoO2-Cu-C electrode is a propitious next-generation anode for LIBs.
ABSTRACT
Water harvesting using the metal-organic framework (MOF)-801 is restricted by limited working capacity, powder structuring, and finite stability. To overcome these issues, MOF-801 is crystallized on the surface of macroporous poly(N-isopropylacrylamide-glycidyl methacrylate) spheres, called P(NIPAM-GMA), through an in situ confined growth strategy, forming spherical MOF-801@P(NIPAM-GMA) composite with temperature-responsive function. By lowering the nucleation energy barrier, the average size of the MOF-801 crystals decreases by 20 times. Thus, abundant defects as adsorption sites for water can be installed in the crystals lattices. As a consequence, the composite provides an unprecedented high water harvesting efficiency. The composite is produced in the kilogram-scale and can capture 1.60 kg H2 O/kg composite/day from 20% relative humidity between 25 and 85 °C. This study provides an effective methodology for improving the adsorption capacity through controlled defects formation as adsorption sites and to improve the kinetics through the design of a composite with macroporous transport channel network.
ABSTRACT
Simultaneously improving the energy density and power density of electrochemical energy storage systems is the ultimate goal of electrochemical energy storage technology. An effective strategy to achieve this goal is to take advantage of the high capacity and rapid kinetics of electrochemical proton storage to break through the power limit of batteries and the energy limit of capacitors. This article aims to review the research progress on the physicochemical properties, electrochemical performance, and reaction mechanisms of electrode materials for electrochemical proton storage. According to the different charge storage mechanisms, the surface redox, intercalation, and conversion materials are classified and introduced in detail, where the influence of crystal water and other nanostructures on the migration kinetics of protons is clarified. Several reported advanced full cell devices are summarized to promote the commercialization of electrochemical proton storage. Finally, this review provides a framework for research directions of charge storage mechanism, basic principles of material structure design, construction strategies of full cell device, and goals of practical application for electrochemical proton storage.
ABSTRACT
Fluorescently labeled, solute-binding proteins that change their fluorescent output in response to ligand binding are frequently used as biosensors for a wide range of applications. We have previously developed a "Computational Identification of Non-disruptive Conjugation sites" (CINC) approach, an in silico pipeline utilizing molecular dynamics simulations for the rapid design and construction of novel protein-fluorophore conjugate-type biosensors. Here, we report an improved in silico scoring algorithm for use in CINC and its use in the construction of an oligogalacturonide-detecting biosensor set. Using both 4,5-unsaturated and saturated oligogalacturonides, we demonstrate that signal transmission from the ligand-binding pocket of the starting protein scaffold to the CINC-selected reporter positions is effective for multiple different ligands. The utility of an oligogalacturonide-detecting biosensor is shown in Carbohydrate Active Enzyme (CAZyme) activity assays, where the biosensor is used to follow product release upon polygalacturonic acid (PGA) depolymerization in real time. The oligogalacturonide-detecting biosensor set represents a novel enabling tool integral to our rapidly expanding platform for biosensor-based carbohydrate detection, and moving forward, the CINC pipeline will continue to enable the rational design of biomolecular tools to detect additional chemically distinct oligosaccharides and other solutes.
Subject(s)
Biosensing Techniques , Fluorescent Dyes , Ligands , Oligosaccharides , ProteinsABSTRACT
In this work, the rapid unfolding kinetics of pancreas α-amylase (PPA) induced by l-arginine and the interaction mechanism were investigated. The unfolding followed a first-level reaction kinetics equation, without intermediates. l-arginine interacted with PPA though diffusion-controlled process rather than complexion. The interaction between l-arginine and PPA resulted in a pronounced decrease in ß-sheet and a significant increase in random coil, and thereby the enzyme activity decreased. However, the unfolding of PPA could be compensated and the second structure change could be recovered to some extent by the macromolecular crowded medium of Pluronics. Further insight into the mechanism disclosed that the broken H-bond network of water may contribute to PPA unfolding. This work provides a new perspective on the interaction of l-arginine with digestive enzyme. The unfolding mechanism of enzymes by may help to understand the effects of other structurally similar drugs, which is of concern in food-drug interactions.
Subject(s)
Pancreatic alpha-Amylases , alpha-Amylases , Amylases , Arginine , Kinetics , alpha-Amylases/metabolismABSTRACT
Fluorescently-labeled solute-binding proteins that alter their fluorescence output in response to ligand binding have been utilized as biosensors for a variety of applications. Coupling protein ligand binding to altered fluorescence output often requires trial and error-based testing of both multiple labeling positions and fluorophores to produce a functional biosensor with the desired properties. This approach is laborious and can lead to reduced ligand binding affinity or altered ligand specificity. Here we report the Computational Identification of Non-disruptive Conjugation sites (CINC) for streamlined identification of fluorophore conjugation sites. By exploiting the structural dynamics properties of proteins, CINC identifies positions where conjugation of a fluorophore results in a fluorescence change upon ligand binding without disrupting protein function. We show that a CINC-developed maltooligosaccharide (MOS)-detecting biosensor is capable of rapid (kon = 20 µM-1s-1), sensitive (sub-µM KD) and selective MOS detection. The MOS-detecting biosensor is modular with respect to the spectroscopic properties and demonstrates portability to detecting MOS released via α-amylase-catalyzed depolymerization of starch using both a stopped-flow and a microplate reader assay. Our MOS-detecting biosensor represents a first-in-class probe whose design was guided by changes in localized dynamics of individual amino acid positions, supporting expansion of the CINC pipeline as an indispensable tool for a wide range of protein engineering applications.
Subject(s)
Biosensing Techniques , Carbohydrates , Fluorescent Dyes , Ligands , Spectrometry, FluorescenceABSTRACT
Calcium (Ca2+) signaling represents a universal information code in plants, playing crucial roles spanning developmental processes to stress responses. Ca2+ signals are decoded into defined plant adaptive responses by different Ca2+ sensing proteins, including calmodulin (CaM) and calmodulin-like (CML) proteins. Although major advances have been achieved in describing how these Ca2+ decoding proteins interact and regulate downstream target effectors, the molecular details of these processes remain largely unknown. Herein, the kinetics of Ca2+ dissociation from a conserved CaM and two CML isoforms from A. thaliana has been studied by fluorescence stopped-flow spectroscopy. Kinetic data were obtained for the isolated Ca2+-bound proteins as well as for the proteins complexed with different target peptides. Moreover, the lobe specific interactions between the Ca2+ sensing proteins and their targets were characterized by using a panel of protein mutants deficient in Ca2+ binding at the N-lobe or C-lobe. Results were analyzed and discussed in the context of the Ca2+-decoding and Ca2+-controlled target binding mechanisms in plants.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Calcium/metabolism , Calmodulin/metabolism , Peptides/metabolism , Arabidopsis/drug effects , Kinetics , Melitten/pharmacology , Mutant Proteins/metabolismABSTRACT
SLC6A14 (solute carrier family 6 member 14) is an amino acid transporter, driven by Na+ and Cl- co-transport, whose structure, function, and molecular and kinetic mechanism have not been well characterized. Its broad substrate selectivity, including neutral and cationic amino acids, differentiates it from other SLC6 family members, and its proposed involvement in nutrient transport in several cancers suggest that it could become an important drug target. In the present study, we investigated SLC6A14 function and its kinetic mechanism after expression in human embryonic kidney (HEK293) cells, including substrate specificity and voltage dependence under various ionic conditions. We applied rapid solution exchange, voltage jumps, and laser photolysis of caged alanine, allowing sub-millisecond temporal resolution, to study SLC6A14 steady state and pre-steady state kinetics. The results highlight the broad substrate specificity and suggest that extracellular chloride enhances substrate transport but is not required for transport. As in other SLC6 family members, Na+ binding to the substrate-free transporter (or conformational changes associated with it) is electrogenic and is likely rate limiting for transporter turnover. Transient current decaying with a time constant of <1ms is also observed after rapid amino acid application, both in forward transport and homoexchange modes, indicating a slightly electrogenic, but fast and not rate-limiting substrate translocation step. Our results, which are consistent with kinetic modeling, suggest rapid transporter turnover rate and substrate translocation with faster kinetics compared with other SLC6 family members. Together, these results provided novel information on the SLC6A14 transport cycle and mechanism, expanding our understanding of SLC6A14 function.
ABSTRACT
The growth of the polypeptide chain occurs due to the fast and coordinated work of the ribosome and protein elongation factors, EF-Tu and EF-G. However, the exact contribution of each of these components in the overall balance of translation kinetics remains not fully understood. We created an in vitro translation system Escherichia coli replacing either elongation factor with heterologous thermophilic protein from Thermus thermophilus. The rates of the A-site binding and decoding reactions decreased an order of magnitude in the presence of thermophilic EF-Tu, indicating that the kinetics of aminoacyl-tRNA delivery depends on the properties of the elongation factor. On the contrary, thermophilic EF-G demonstrated the same translocation kinetics as a mesophilic protein. Effects of translocation inhibitors (spectinomycin, hygromycin B, viomycin and streptomycin) were also similar for both proteins. Thus, the process of translocation largely relies on the interaction of tRNAs and the ribosome and can be efficiently catalysed by thermophilic EF-G even at suboptimal temperatures.
Subject(s)
Bacterial Proteins/metabolism , Escherichia coli/metabolism , Peptide Chain Elongation, Translational , Peptide Elongation Factor Tu/metabolism , Ribosomes/metabolism , Thermus thermophilus , Peptide Elongation Factor G/metabolism , RNA, Bacterial/metabolism , RNA, Transfer/metabolismABSTRACT
The presence of excess fluoride (F- > 1.5 mg/L) in drinking water affects more than 260 million people globally and leads to dental and skeletal fluorosis among other health problems. This study investigated fluoride removal by graphene oxide-ceria nanohybrid (GO-CeO2) and elucidated the mechanisms involved. The nanohybrid exhibited ultra-rapid kinetics for fluoride removal and the equilibrium (85% removal, 10 mg F-/L initial concentration) was achieved within 1 min which is one of the fastest kinetics for fluoride removal reported so far. Fluoride removal by the nanohybrid followed Langmuir isotherm with a maximum adsorption capacity of 8.61 mg/g at pH 6.5 and that increased to 16.07 mg/g when the pH was lowered to 4.0. Based on the experimental results and characterization data, we have postulated that both electrostatic interaction and surface complexation participated in the fluoride removal process. The O2- ions present in the CeO2 lattice were replaced by F- ions to make a coordination compound (complex). While both Ce4+ and Ce3+ were present in ceria nanoparticles (CeO2 NPs), Ce3+ participated in fluoride complexation. During fluoride removal by GO-CeO2, the GO sheets acted as electron mediators and help to reduce Ce4+ to Ce3+ at the CeO2 NPs-GO interface, and the additional Ce3+ enhanced fluoride removal by the nanohybrid.
Subject(s)
Drinking Water , Nanoparticles , Water Pollutants, Chemical , Water Purification , Adsorption , Fluorides , Humans , Hydrogen-Ion Concentration , Kinetics , Water Pollutants, Chemical/analysisABSTRACT
Many antibiotics that bind to the ribosome inhibit translation by blocking the movement of tRNAs and mRNA or interfering with ribosome dynamics, which impairs the formation of essential translocation intermediates. Here we show how translocation inhibitors viomycin (Vio), neomycin (Neo), paromomycin (Par), kanamycin (Kan), spectinomycin (Spc), hygromycin B (HygB), and streptomycin (Str, an antibiotic that does not inhibit tRNA movement), affect principal motions of the small ribosomal subunits (SSU) during EF-G-promoted translocation. Using ensemble kinetics, we studied the SSU body domain rotation and SSU head domain swiveling in real time. We show that although antibiotics binding to the ribosome can favor a particular ribosome conformation in the absence of EF-G, their kinetic effect on the EF-G-induced transition to the rotated/swiveled state of the SSU is moderate. The antibiotics mostly inhibit backward movements of the SSU body and/or the head domains. Vio, Spc, and high concentrations of Neo completely inhibit the backward movements of the SSU body and head domain. Kan, Par, HygB, and low concentrations of Neo slow down both movements, but their sequence and coordination are retained. Finally, Str has very little effect on the backward rotation of the SSU body domain, but retards the SSU head movement. The data underscore the importance of ribosome dynamics for tRNA-mRNA translocation and provide new insights into the mechanism of antibiotic action.
Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli/drug effects , Protein Biosynthesis/drug effects , RNA, Messenger/metabolism , RNA, Transfer/metabolism , Ribosome Subunits/drug effects , Biological Transport , Cinnamates/pharmacology , Escherichia coli/genetics , Escherichia coli/metabolism , Hygromycin B/analogs & derivatives , Hygromycin B/pharmacology , Kanamycin/pharmacology , Kinetics , Neomycin/pharmacology , Paromomycin/pharmacology , Peptide Elongation Factor G/genetics , Peptide Elongation Factor G/metabolism , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Transfer/antagonists & inhibitors , RNA, Transfer/chemistry , RNA, Transfer/genetics , Ribosome Subunits/genetics , Ribosome Subunits/metabolism , Ribosome Subunits/ultrastructure , Spectinomycin/pharmacology , Streptomycin/pharmacology , Viomycin/pharmacologyABSTRACT
Stalled DNA replication forks can result in incompletely replicated genomes and cell death. DNA replication restart pathways have evolved to deal with repair of stalled forks and E. coli Rep helicase functions in this capacity. Rep and an accessory protein, PriC, assemble at a stalled replication fork to facilitate loading of other replication proteins. A Rep monomer is a rapid and processive single stranded (ss) DNA translocase but needs to be activated to function as a helicase. Activation of Rep in vitro requires self-assembly to form a dimer, removal of its auto-inhibitory 2B sub-domain, or interactions with an accessory protein. Rep helicase activity has been shown to be stimulated by PriC, although the mechanism of activation is not clear. Using stopped flow kinetics, analytical sedimentation and single molecule fluorescence methods, we show that a PriC dimer activates the Rep monomer helicase and can also stimulate the Rep dimer helicase. We show that PriC can self-assemble to form dimers and tetramers and that Rep and PriC interact in the absence of DNA. We further show that PriC serves as a Rep processivity factor, presumably co-translocating with Rep during DNA unwinding. Activation is specific for Rep since PriC does not activate the UvrD helicase. Interaction of PriC with the C-terminal acidic tip of the ssDNA binding protein, SSB, eliminates Rep activation by stabilizing the PriC monomer. This suggests a likely mechanism for Rep activation by PriC at a stalled replication fork.
Subject(s)
DNA Helicases/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , DNA, Bacterial/metabolism , DNA, Single-Stranded/metabolism , Escherichia coli/chemistry , Gene Expression Regulation, Bacterial , Microscopy, Fluorescence , Models, Molecular , Protein Binding , Protein Conformation , Protein Multimerization , Single Molecule ImagingABSTRACT
Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami (G542V, G581A, or ins544V) leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami.
ABSTRACT
Electron bifurcation uses free energy from exergonic redox reactions to power endergonic reactions. ß-FAD of the electron transfer flavoprotein (EtfAB) from the anaerobic bacterium Acidaminococcus fermentans bifurcates the electrons of NADH, sending one to the low-potential ferredoxin and the other to the high-potential α-FAD semiquinone (α-FADâ¢-). The resultant α-FAD hydroquinone (α-FADH-) transfers one electron further to butyryl-CoA dehydrogenase (Bcd); two such transfers enable Bcd to reduce crotonyl-CoA to butyryl-CoA. To get insight into the mechanism of these intricate reactions, we constructed an artificial reaction only with EtfAB containing α-FAD or α-FADâ¢- to monitor formation of α-FADâ¢- or α-FADH-, respectively, using stopped flow kinetic measurements. In the presence of α-FAD, we observed that NADH transferred a hydride to ß-FAD at a rate of 920 s-1, yielding the charge-transfer complex NAD+:ß-FADH- with an absorbance maximum at 650 nm. ß-FADH- bifurcated one electron to α-FAD and the other electron to α-FAD of a second EtfAB molecule, forming two stable α-FADâ¢-. With α-FADâ¢-, the reduction of ß-FAD with NADH was 1500 times slower. Reduction of ß-FAD in the presence of α-FAD displayed a normal kinetic isotope effect (KIE) of 2.1, whereas the KIE was inverted in the presence of α-FADâ¢-. These data indicate that a nearby radical (14 Å apart) slows the rate of a hydride transfer and inverts the KIE. This unanticipated flavin chemistry is not restricted to Etf-Bcd but certainly occurs in other bifurcating Etfs found in anaerobic bacteria and archaea.
Subject(s)
Acidaminococcus/metabolism , Bacterial Proteins/metabolism , Electron-Transferring Flavoproteins/metabolism , Flavins/metabolism , Electron Transport , Kinetics , Oxidation-Reduction , PhylogenyABSTRACT
Sodium-selenium (Na-Se) battery has been emerging as one of the most prospective energy storage systems owing to their high volumetric energy density and cost effectiveness. Nevertheless, the shuttle effect of sodium polyselenide (NaPSe) and sluggish electrochemical reaction kinetics present the main bottlenecks for its practical implementation. Herein, a new Se host of 3D nitrogen-doped hierarchical multicavity carbon nanospheres (3D NHMCs) is designed and synthesized via a facile self-sacrifice templating strategy. The 3D NHMCs are verified to hold a favorable structure of a hollow macropore core and numerous micro/mesopores hollow shell for hosting Se, which can not only maximize Se utilization and alleviate the volumetric expansion but also promote the electrical/ionic conductivity and electrolyte infiltration. Moreover, the abundant self-functionalized surfaces as an efficient NaPSe scavenger via robust physical-chemical dual blocking effects demonstrate high-efficiency in situ anchoring-diffusion-conversion of NaPSe, rendering rapid reaction kinetics and remarkable suppressive shuttle effect, as evidenced by systematic experimental analysis and density functional theory calculations. As a result, the high-Se-loading 3D NHMCs/Se cathode exhibits an ultrahigh volumetric capacity (863 mAh cm-3 ) and rate capability (377 mAh g-1 at 20 C) and unexceptionable stability over 2000 cycles at 2 C.
ABSTRACT
The chapter focuses on the methods involved in producing and characterizing two key nickel-iron-sulfur enzymes in the Wood-Ljungdahl pathway (WLP) of anaerobic conversion of carbon dioxide fixation into acetyl-CoA: carbon monoxide dehydrogenase (CODH) and acetyl-CoA synthase (ACS). The WLP is used for biosynthesis of cell material and energy conservation by anaerobic bacteria and archaea, and it is central to several industrial biotechnology processes aimed at using syngas and waste gases for the production of fuels and chemicals. The pathway can run in reverse to allow organisms, e. g., methanogens and sulfate reducers, to grow on acetate. The CODH and ACS intertwine to form a tenacious CODH/ACS complex that converts CO2, a methyl group, and coenzyme A into acetyl-CoA. CODH also behaves as a modular unit that can function as an independent homodimer. Besides coupling to ACS, CODH can interact with hydrogenases to couple CO oxidation to H2 formation. These enzymes have been purified and characterized from several microbes.