Low-potential Reductants Research Articles

Low Potential CO2 Reduction by Inert Fe(II)-Macrobicyclic Complex: A New Concept of Cavity Assisted CO2 Activation.

The advantage of a pre-organized π-cavity of Fe(II) complex of a newly developed macrobicycle cryptand is explored for CO2 reduction by overcoming the problem of high overpotential associated with the inert nature of the cryptate. Thus, a bipyridine-centered tritopic macrobicycle having a molecular π-cavity capable of forming Fe(II) complex as well as potential for CO2 encapsulation is synthesized. The inert Fe(II)-cryptate shows much lower potential in cyclic voltammetry than the Fe(II)-tris-dimethylbipyridine (Fe-MBP) core. Interestingly, this cryptate shows electrochemical CO2 reduction at a considerably lower potential than the Fe-MBP inert core. Therefore, this study represents that a well-structured π-cavity may generate a new series of molecular catalysts for the CO2 reduction reaction (CO2 RR), even with the inert metal complexes.

Anaerobic <i>Thermodesulfovibrio</i> and Aerobic <i>Meiothermus</i> Coexist in Deep Thermal Water

Abstract—Research on the microorganisms inhabiting deep aquifers is based on sampling the water released from deep wells and is seldom concerned with the physicochemical processes of the water-rock system. The issue of metabolism of aerobic prokaryotes revealed in deep habitats by molecular techniques remains unclear. Cultivation is required for direct determination of relation of prokaryotes to oxygen. In the present work, aerobic and anaerobic bacteria, which were revealed in thermal radon baths of the Belokurikha resort by molecular techniques, were isolated. Profiling by the 16S rRNA gene revealed predominance of members of the Deionococcus-Thermus group belonging to the genus Meiothermus (17.6% reads) and considered strictly aerobic. Anaerobic sulfate-reducing Thermodesulfovibrio were also present in the sample. The habitat was characterized by reductive, alkaline conditions. Target-oriented cultivation revealed aerobically growing Meiothermus sp. 1165, which was closely related to Meiothermus cerbereus. An alkaliphilic anaerobic sulfate reducer Thermodesulfovibrio sp. 1176 was also isolated. The rate of sulfate reduction measured in the Belokurikha water using \({\text{Na}}_{{\text{2}}}^{{{\text{ 35}}}}{\text{S}}{{{\text{O}}}_{{\text{4}}}}\) yielded the value of 41.4 ± 1.06 µm Sred L–1 day–1, or 1.29 nmol S mL–1 day–1. Analysis of the genome of strain 1176 revealed the presence of various mechanisms responsible for its relative resistance to oxygen and oxidative stress, which included superoxide reductase, rubredoxin, a Fe-Mn family superoxide dismutase, a KatG catalase-peroxidase, and a cytochrome bd ubiquinol oxidase. The low redox potential and intense anaerobic sulfate reduction provide evidence for the generally reduced conditions in the Belokurikha deep horizons. Spatial separation of aerobes and anaerobes in the water-rock system, similar to the one occurring in the terrestrial microbial mats, may be hypothesized, as well as occurring of aerobic processes in microniches.

Computational description of surface hydride phases on Pt(111) electrodes.

Surface platinum hydride structures may exist and play a potentially important role during electrocatalysis and cathodic corrosion of Pt(111). Earlier work on platinum hydrides suggests that Pt may form clusters with multiple equivalents of hydrogen. Here, using thermodynamic methods and density functional theory, we compared several surface hydride structures on Pt(111). The structures contain multiple monolayers of hydrogen in or near the surface Pt layer. The hydrogen in these structures may bind the subsurface or reconstruct the surface both in the set of initial configurations and in the resulting (meta)stable structures. Multilayer stable configurations share one monolayer of subsurface H stacking between the top two Pt layers. The structure containing two monolayers (MLs) of H is formed at -0.29V vs normal hydrogen electrode, is locally stable with respect to configurations with similar H densities, and binds H neutrally. Structures with 3 and 4 ML H form at -0.36 and -0.44V, respectively, which correspond reasonably well to the experimental onset potential of cathodic corrosion on Pt(111). For the 3 ML configuration, the top Pt layer is reconstructed by interstitial H atoms to form a well-ordered structure with Pt atoms surrounded by four, five, or six H atoms in roughly square-planar and octahedral coordination patterns. Our work provides insight into the operando surface state during low-potential reduction reactions on Pt(111) and shows a plausible precursor for cathodic corrosion.

Electrochemical Active MOF Nanoparticles/Carbon Composite for the Electroreduction of CO2 into Valuable Chemicals

The present work suggests the effectiveness of the composition method to assist non-conductive MOFs to participate in electrochemical applications. We propose a new approach to grow MOF nanoparticles into conductive porous carbon–HKUST-1. When operated in a CO2-saturated aqueous solution, the composites exhibit excellent electrochemical performances, including a low onset potential (-0.58 V) and large reduction currents (-17 mA.cm-2 at -1.0 V), which is considerably high compared to usually reported MOF-based beyond CO electroreduction. The strong π- π interaction between the aromatic linkers and the graphitic carbon proved advantageous in inducing high lateral conductivity of up to 17.2 S m-1 to the composite. EPR investigation of the unpaired electrons on the Cu atoms also disclose that the induced conductivity influences the metal centers. Electrochemical studies reveal high electroactive coverage of 155 nmol. Besides these factors, the excellent durability of the composite is worth noticing. The composite stabilizes the active material, and after 12 hours of constant operation, the electrochemical response was the same. Most importantly, the composite produces formic acid, a valuable chemical commodity, within a short time interval of 15 minutes.

Image texture, low contrast liver lesion detectability and impact on dose: Deep learning algorithm compared to partial model-based iterative reconstruction

ObjectivesTo compare deep learning (True Fidelity, TF) and partial model based Iterative Reconstruction (ASiR-V) algorithm for image texture, low contrast lesion detectability and potential dose reduction. MethodsAnthropomorphic phantoms (mimicking non-overweight and overweight patient), containing lesions of 6 mm in diameter with 20HU contrast, were scanned at five different dose levels (2,6,10,15,20 mGy) on a CT system, using clinical routine protocols for liver lesion detection. Images were reconstructed using ASiR-V 0% (surrogate for FBP), 60 % and TF at low, medium and high strength. Noise texture was characterized by computing a normalized Noise Power Spectrum filtered by an eye filter. The similarity against FBP texture was evaluated using peak frequency difference (PFD) and root mean square deviation (RMSD). Low contrast detectability was assessed using a channelized Hotelling observer and the area under the ROC curve (AUC) was used as figure of merit. Potential dose reduction was calculated to obtain the same AUC for TF and ASiR-V. ResultsFBP-like noise texture was more preserved with TF (PFD from -0.043mm-1 to -0.09mm-1, RMSD from 0.12mm-1 to 0.21mm-1) than with ASiR-V (PFD equal to 0.12 mm-1, RMSD equal to 0.53mm-1), resulting in a sharper image. AUC was always higher with TF than ASIR-V. In average, TF compared to ASiR-V, enabled a radiation dose reduction potential of 7%, 25 % and 33 % for low, medium and high strength respectively. ConclusionCompared to ASIR-V, TF at high strength does not impact noise texture and maintains low contrast liver lesions detectability at significant lower dose.

Direct left ventricular wire pacing during transcatheter aortic valve implantation.

Rapid ventricular pacing is used during balloon aortic valvuloplasty, balloon‑expandable transcatheter aortic valve implantation (TAVI), and for postdilatation. Right ventricular (RV) lead pacing has been regarded as a gold standard. Direct left ventricular (LV) wire pacing has recently been considered safe and effective in TAVI interventions. This study aimed to analyze procedural outcomes of direct LV pacing compared with RV stimulation in unselected patients undergoing TAVI. Direct LV wire pacing was provided via available preshaped guidewires and used only when no predictors of atrioventricular block were present. The primary study objective was the assessment of the efficacy of direct LV wire pacing. The secondary objectives included the evaluation of procedure duration and safety in comparison with the conventional method. A combined endpoint (major adverse cardiovascular event) was defined as the occurrence of death, stroke, venous puncture-related complications, and cardiac tamponade. In 2017 and 2018, 143 patients underwent transfemoral TAVI. Of these, 114 (79.7%) had self‑ ‑expandable valves implanted. Direct LV wire pacing was the dominant method of pacing (82 patients [57.3%]), and its efficacy reached 97.6%. The median (interquartile range) procedure time was shorter in the direct LV wire pacing group (80 [70-90] min vs 85 [70-95] min; P = 0.02). Major adverse cardiovascular events were more frequent in the RV lead pacing group (11.5% vs 4.9%), but no statistical significance was achieved (P = 0.13). Direct LV wire pacing during TAVI is a simple, reproducible, and safe technique, which provides reliable, sustained stimulation with a low complication rate and potential reduction of procedural time.

Responses of Total Biomass, Shoot Dry Weight, Yield and Yield Components of Jerusalem Artichoke (Helianthus tuberosus L.) Varieties under Different Terminal Drought Duration

As a secondary crop planting in the double-cropping system, terminal drought is the major constraint of Jerusalem artichoke production in the rainfed area. This study aims to estimate the effects of different durations of terminal drought on total biomass, tuber yields, harvest index, yield components, and to identify high yield potential and low yield reduction genotypes for the varietal improvement program. A split-plot design with four replications was conducted under field experiment for two years. Three drought durations including non-drought stress (SD0), drought from 60 and 45 days after transplanting until harvest (SD1 and SD2, respectively), were assigned in main plots and six genotypes were arranged in subplots. Crop parameters were greater decreased under a long-drought duration than under short-drought duration. The genotypes were identified; HEL256, JA37 and JA125 had high yield potential under SD0 conditions, whereas there was high yield reduction under drought conditions. In contrast, JA60 and HEL253 were identified as low yield potential and low yield reduction genotypes. This information suggested that high yield potential genotypes and low yield reduction genotypes should be selected and generated progeny population for improvement of new varieties with high yield potential and low yield reduction for growing in terminal drought-prone environments.

Electron Paramagnetic Resonance Spectroscopic Identification of the Fe-S Clusters in the SPASM Domain-Containing Radical SAM Enzyme PqqE.

Pyrroloquinoline quinone (PQQ) is an important redox active quinocofactor produced by a wide variety of bacteria. A key step in PQQ biosynthesis is a carbon-carbon cross-link reaction between glutamate and tyrosine side chains within the ribosomally synthesized peptide substrate PqqA. This reaction is catalyzed by the radical SAM enzyme PqqE. Previous X-ray crystallographic and spectroscopic studies suggested that PqqE, like the other members of the SPASM domain family, contains two auxiliary Fe-S clusters (AuxI and AuxII) in addition to the radical SAM [4Fe-4S] cluster. However, a clear assignment of the electron paramagnetic resonance (EPR) signal of each Fe-S cluster was hindered by the isolation of a His6-tagged PqqE variant with an altered AuxI cluster. In this work, we are able to isolate soluble PqqE variants by using a less disruptive strep-tactin chromatographic approach. We have unambiguously identified the EPR signatures for four forms of Fe-S clusters present in PqqE through the use of multifrequency EPR spectroscopy: the RS [4Fe-4S] cluster, the AuxII [4Fe-4S] cluster, and two different clusters ([4Fe-4S] and [2Fe-2S]) bound in the AuxI site. The RS [4Fe-4S] cluster, the AuxII [4Fe-4S] cluster, and the [2Fe-2S] cluster form in the AuxI site can all be reduced by sodium dithionite, with g tensors of their reduced form determined as [2.040, 1.927, 1.897], [2.059, 1.940, 1.903], and [2.004, 1.958, 1.904], respectively. The AuxI [4Fe-4S] cluster that is determined on the basis of its relaxation profile can be reduced only by using low-potential reductants such as Ti(III) citrate or Eu(II)-DTPA to give rise to a g1 = 2.104 signal. Identification of the EPR signature for each cluster paves the way for further investigations of SPASM domain radical SAM enzymes.

Low-potential reduction of sulfite at a ruthenium-oxide hexacyanoferrate modified electrode

The low-potential reduction of sulfite at a ruthenium-oxide hexacyanoferrate (RuOHCF)-modified electrode is reported. A decrease (200mV) in the overpotential of the sulfite reduction reaction and a substantial current increase (compared to the bare glassy-carbon electrode) were verified. Evidence is found for a mass transport regime which includes a thin-layer diffusional process (sulfite species trapped within the RuOHCF film) responsible for the lowered reductive potential (rather than electrocatalytic properties of the film). The low-potential (−0.2V vs. Ag/AgCl/saturated KCl) amperometric detection of sulfite in 0.25molL−1 HCl was free of interferences from substances typically found in food samples.

Spectroscopic and crystallographic characterization of "alternative resting" and "resting oxidized" enzyme forms of bilirubin oxidase: implications for activity and electrochemical behavior of multicopper oxidases.

While there is broad agreement on the catalytic mechanism of multicopper oxidases (MCOs), the geometric and electronic structures of the resting trinuclear Cu cluster have been variable, and their relevance to catalysis has been debated. Here, we present a spectroscopic characterization, complemented by crystallographic data, of two resting forms occurring in the same enzyme and define their interconversion. The resting oxidized form shows similar features to the resting form in Rhus vernicifera and Trametes versicolor laccase, characterized by "normal" type 2 Cu electron paramagnetic resonance (EPR) features, 330 nm absorption shoulder, and a short type 3 (T3) Cu-Cu distance, while the alternative resting form shows unusually small A(||) and high g(||) EPR features, lack of 330 nm absorption intensity, and a long T3 Cu-Cu distance. These different forms are evaluated with respect to activation for catalysis, and it is shown that the alternative resting form can only be activated by low-potential reduction, in contrast to the resting oxidized form which is activated via type 1 Cu at high potential. This difference in activity is correlated to differences in redox states of the two forms and highlights the requirement for efficient sequential reduction of resting MCOs for their involvement in catalysis.

Uncoupling Nitrogenase: Catalytic Reduction of Hydrazine to Ammonia by a MoFe Protein in the Absence of Fe Protein-ATP

The catalytic reduction of hydrazine (N(2)H(4)) to ammonia by a β-98(Tyr→His) MoFe protein in the absence of the Fe protein or ATP is reported. The reduction of N(2) or other substrates (e.g., hydrazine, protons, acetylene) by nitrogenase normally requires the transient association of the two nitrogenase component proteins, the Fe protein and the MoFe protein. The Fe protein, with two bound MgATP molecules, transfers one electron to the MoFe protein during each association, coupled to the hydrolysis of two MgATP. All substrate reduction reactions catalyzed by nitrogenase require delivery of electrons by the Fe protein coupled to the hydrolysis of MgATP. We report that when a single amino acid within the MoFe protein (β-98(Tyr)) is substituted by His, the resulting MoFe protein supports catalytic reduction of the nitrogenous substrate hydrazine (N(2)H(4)) to two ammonia molecules when provided with a low potential reductant, polyaminocarboxylate ligated Eu(II) (E(m) -1.1 V vs NHE). The wild-type and a number of other MoFe proteins with amino acid substitutions do not show significant rates of hydrazine reduction under these conditions, whereas the β-98(His) MoFe protein catalyzes hydrazine reduction at rates up to 170 nmol NH(3)/min/mg MoFe protein. This rate of hydrazine reduction is 94% of the rate catalyzed by the β-98(His) or wild-type MoFe protein when combined with the Fe protein, ATP, and reductant under comparable conditions. The β-98(His) MoFe protein reduction of hydrazine in the absence of the Fe protein showed saturation kinetics for the concentration of reductant and substrate. The implications of these results in understanding the nitrogenase mechanism are discussed.

Characterization of energy-conserving hydrogenase B in Methanococcus maripaludis.

The Methanococcus maripaludis energy-conserving hydrogenase B (Ehb) generates low potential electrons required for autotrophic CO(2) assimilation. To analyze the importance of individual subunits in Ehb structure and function, markerless in-frame deletions were constructed in a number of M. maripaludis ehb genes. These genes encode the large and small hydrogenase subunits (ehbN and ehbM, respectively), a polyferredoxin and ferredoxin (ehbK and ehbL, respectively), and an ion translocator (ehbF). In addition, a gene replacement mutation was constructed for a gene encoding a putative membrane-spanning subunit (ehbO). When grown in minimal medium plus acetate (McA), all ehb mutants had severe growth deficiencies except the DeltaehbO::pac strain. The membrane-spanning ion translocator (DeltaehbF) and the large hydrogenase subunit (DeltaehbN) deletion strains displayed the severest growth defects. Deletion of the ehbN gene was of particular interest because this gene was not contiguous to the ehb operon. In-gel activity assays and Western blots confirmed that EhbN was part of the membrane-bound Ehb hydrogenase complex. The DeltaehbN strain was also sensitive to growth inhibition by aryl acids, indicating that Ehb was coupled to the indolepyruvate oxidoreductase (Ior), further supporting the hypothesis that Ehb provides low potential reductants for the anabolic oxidoreductases in M. maripaludis.

Reduction of the Iron−Sulfur Clusters in Mitochondrial NADH:Ubiquinone Oxidoreductase (Complex I) by EuII-DTPA, a Very Low Potential Reductant

NADH:ubiquinone oxidoreductase (complex I) is the first enzyme of the mitochondrial electron transport chain. It contains a flavin mononucleotide to oxidize NADH, and eight iron-sulfur clusters. Seven of them transfer electrons between the flavin and the quinone-binding site, and one is on the opposite side of the flavin. Although most information about their properties is from EPR, the spectra from only five clusters have been observed, and it is difficult to match them to the structurally defined clusters. Here, we analyze complex I from bovine mitochondria reacted with a very low potential reductant, to impose a potential approaching -1 V. We compare the spectra with those from higher potentials and from the 24 kDa subunit and flavoprotein subcomplex, and model the spectra by starting from those with fewer components and building the complexity gradually. Spectrum N1a, from the 24 kDa subunit [2Fe-2S] cluster, is not observed in bovine complex I at any potential. Spectrum N1b, from the 75 kDa subunit [2Fe-2S] cluster, exhibits a lower potential than the N3, N4 and N5 spectra of three [4Fe-4S] clusters. In the lowest potential spectra an N5-type spectrum is observed at unusually high temperature (indicating a significant change to the cluster, or that two clusters have very similar g values), the relaxation rate of N1b increases (indicating that a nearby cluster has become reduced) and a new feature with an apparent g value of 2.16 suggests an interaction between two reduced clusters. The consequences of these observations for electron transfer in complex I are discussed.

Mössbauer Evidence for an Exchange-Coupled {[Fe4S4]1+ Nip1+} A-Cluster in Isolated α Subunits of Acetyl-Coenzyme A Synthase/Carbon Monoxide Dehydrogenase

The active site A-cluster in the alpha subunit of the title enzyme consists of an Fe4S4 cluster coordinated to a [Nip Nid] subcomponent. The cluster must be activated for catalysis using low-potential reductants such as Ti(III) citrate. Relative to the inactive {[Fe4S4]2+ Nip2+ Nid2+} state, the activated state appears to be 2-electrons more reduced, but the location of these electrons within the A-cluster is uncertain, with {[Fe4S4]2+ Nip0 Nid2+} and {[Fe4S4]1+ Nip1+ Nid2+} configurations proposed. Recombinant apo-alpha subunits oligomerize after activation with NiCl2. The dimer fraction, upon reduction with excess Ti(III)citrate, exhibited Mössbauer spectra consisting of two quadrupole doublets representing 51% and 21% of the Fe, with parameters indicating [Fe4S4]1+ states. Spectra recorded in strong magnetic fields were typical of diamagnetic systems, indicating an exchange-coupled S = 0 {[Fe4S4]1+ Nip1+} state. Additional treatment with CO altered the doublet Mössbauer parameters, suggesting an interaction with CO, but maintaining the cluster in the {[Fe4S4]1+ Nip1+} state. Reduction with substoichiometric equivalents of Ti(III) citrate afforded an EPR signal typical of Ni1+ ions, with g parallel = 2.10 and g perpendicular = 2.02. Addition of more Ti caused the signal intensity to decline, suggesting that it arises from the semireduced {[Fe4S4]2+ Nip1+} state.

Photosystem I/Molecular Wire/Metal Nanoparticle Bioconjugates for the Photocatalytic Production of H2

Photosystem I (PS I) is a robust photosynthetic complex that adeptly captures photons to create a charge-separated state with a quantum efficiency that approaches 1.0. This charge-separated state is stable for approximately 100 ms, and the low-potential reductant that is produced is poised at a redox potential favorable for H2 evolution. PS I has been covalently linked to Pt and Au nanoparticle surfaces by 1,6-hexanedithiol which serves as a molecular wire to both connect PS I to the particles and transfer electrons from the terminal electron transfer cofactor of PS I, FB, to the nanoparticle. Illumination of these Photosystem I/molecular wire/nanoparticle bioconjugates is able to catalyze the reaction: 2H+ + 2e(-)--> H2. Transfer of the electrons from PS I to the nanoparticle through the molecular wire is not rate-limiting for H2 evolution. Supplying the system with more efficient donor-side electron donating species results in a 5-fold increase in the rate of H2 evolution.

A fulleropyrrolidine end-capped platinum-acetylide triad: the mechanism of photoinduced charge transfer in organometallic photovoltaic cells

The fullerene end-capped platinum acetylide donor-acceptor triad Pt(2)ThC(60) was synthesized and characterized by using photophysical methods and photovoltaic device testing. The triad consists of the platinum acetylide oligomer Ph-[triple bond, length as m-dash]-Pt(PBu3)2-[triple bond, length as m-dash]-Th-[triple bond, length as m-dash]-Pt(PBu3)2-[triple bond, length as m-dash]-Ph (Ph=phenyl and Th=2,5-thienyl, stereochemistry at both Pt centers is trans) that contains fulleropyrrolidine moieties on each of the terminal phenylene units. Electrochemistry of the triad reveals relatively low potential oxidation and reduction waves corresponding, respectively, to oxidation of the platinum acetylide and reduction of the fulleropyrrolidine units. Photoluminescence spectroscopy shows that the singlet and triplet states of the platinum acetylide chromophore are strongly quenched in the triad assembly, both in solution at ambient temperature as well as in a low-temperature solvent glass. The excited state quenching arises due to intramolecular photoinduced electron transfer to produce a charge separated state based on charge transfer from the platinum acetylide (donor) to the fulleropyrrolidine (acceptor). Picosecond time resolved absorption spectroscopy confirms that the charge transfer state is produced within 1 ps of photoexcitation, and it decays by charge recombination within 400 ps. Organic photovoltaic devices fabricated using spin-coated films of Pt2ThC60 as the active material operate with modest efficiency, exhibiting a short circuit photocurrent of 0.51 mA cm(-2) and an open circuit voltage of 0.41 V under 100 mW cm(-2)/AM1.5 illumination. The results are discussed in terms of the relationship between the mechanism of photoinduced electron transfer in the triad and the comparatively efficient photovoltaic response exhibited by the material.

The Design of Metal Chelates with a Biologically Related Redox‐Active Part: Conjugation of Riboflavin to Bis(2‐pyridylmethyl)amine Ligand and Preparation of a Ferric Complex

AbstractReaction of a twofold molar excess of bis(2‐pyridylmethyl)amine DPA with 8α‐bromoAc4riboflavin in dry DMF over 24 h affords the 8α‐[bis(2‐pyridylmethyl)amine]‐N‐Ac4riboflavin ligand. This compound can be described as a tweezer in which the flavin moiety acts as a potential electron mediator. The trichloroferric complex has been prepared, and together with the low potential reduction wave of the flavin moiety, the presence of a reversible FeIII/FeII couple at a higher potential is observed. The deacetylated ligand 8α‐[bis(2‐pyridylmethyl)amine]‐N‐riboflavin can easily be obtained. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Similar Transition States Mediate the Q-cycle and Superoxide Production by the Cytochrome bc1 Complex

The cytochrome bc complexes found in mitochondria, chloroplasts and many bacteria play critical roles in their respective electron transport chains. The quinol oxidase (Q(o)) site in this complex oxidizes a hydroquinone (quinol), reducing two one-electron carriers, a low potential cytochrome b heme and the "Rieske" iron-sulfur cluster. The overall electron transfer reactions are coupled to transmembrane translocation of protons via a "Q-cycle" mechanism, which generates proton motive force for ATP synthesis. Since semiquinone intermediates of quinol oxidation are generally highly reactive, one of the key questions in this field is: how does the Q(o) site oxidize quinol without the production of deleterious side reactions including superoxide production? We attempt to test three possible general models to account for this behavior: 1) The Q(o) site semiquinone (or quinol-imidazolate complex) is unstable and thus occurs at a very low steady-state concentration, limiting O(2) reduction; 2) the Q(o) site semiquinone is highly stabilized making it unreactive toward oxygen; and 3) the Q(o) site catalyzes a quantum mechanically coupled two-electron/two-proton transfer without a semiquinone intermediate. Enthalpies of activation were found to be almost identical between the uninhibited Q-cycle and superoxide production in the presence of antimycin A in wild type. This behavior was also preserved in a series of mutants with altered driving forces for quinol oxidation. Overall, the data support models where the rate-limiting step for both Q-cycle and superoxide production is essentially identical, consistent with model 1 but requiring modifications to models 2 and 3.

Genetic construction of truncated and chimeric metalloproteins derived from the alpha subunit of acetyl-CoA synthase from Clostridium thermoaceticum.

In this study, a genetics-based method is used to truncate acetyl-coenzyme A synthase from Clostridium thermoaceticum (ACS), an alpha(2)beta(2) tetrameric 310 kDa bifunctional enzyme. ACS catalyzes the reversible reduction of CO(2) to CO and the synthesis of acetyl-CoA from CO (or CO(2) in the presence of low-potential reductants), CoA, and a methyl group bound to a corrinoid-iron sulfur protein (CoFeSP). ACS contains seven metal-sulfur clusters of four different types called A, B, C, and D. The B, C, and D clusters are located in the 72 kDa beta subunit, while the A-cluster, a Ni-X-Fe(4)S(4) cluster that serves as the active site for acetyl-CoA synthase activity, is located in the 82 kDa alpha subunit. The extent to which the essential properties of the cluster, including catalytic, redox, spectroscopic, and substrate-binding properties, were retained as ACS was progressively truncated was determined. Acetyl-CoA synthase catalytic activity remained when the entire beta subunit was removed, as long as CO, rather than CO(2) and a low-potential reductant, was used as a substrate. Truncating an approximately 30 kDa region from the N-terminus of the alpha subunit yielded a 49 kDa protein that lacked catalytic activity but exhibited A-cluster-like spectroscopic, redox, and CO-binding properties. Further truncation afforded a 23 kDa protein that lacked recognizable A-cluster properties except for UV-vis spectra typical of [Fe(4)S(4)](2+) clusters. Two chimeric proteins were constructed by fusing the gene encoding a ferredoxin from Chromatium vinosum to genes encoding the 49 and 82 kDa fragments of the alpha subunit. The chimeric proteins exhibited EPR signals that were not the simple sum of the signals from the separate proteins, suggesting magnetic interactions between clusters. This study highlights the potential for using genetics to simplify the study of complex multicentered metalloenzymes and to generate new complex metalloenzymes with interesting properties.

Redundancy of aerobic respiratory chains in bacteria? Routes, reasons and regulation

Bacteria are the most remarkable organisms in the biosphere, surviving and growing in environments that support no other life forms. Underlying this ability is a flexible metabolism controlled by a multitude of environmental sensors and regulators of gene expression. It is not surprising, therefore, that bacterial respiration is complex and highly adaptable: virtually all bacteria have multiple, branched pathways for electron transfer from numerous low-potential reductants to several terminal electron acceptors. Such pathways, particularly those involved in anaerobic respiration, may involve periplasmic components, but the respiratory apparatus is largely membrane-bound and organized such that electron flow is coupled to proton (or sodium ion) transport, generating a protonmotive force. It has long been supposed that the multiplicity of pathways serves to provide flexibility in the face of environmental stresses, but the existence of apparently redundant pathways for electrons to a single acceptor, say dioxygen, is harder to explain. Clues have come from studying the expression of oxidases in response to growth conditions, the phenotypes of mutants lacking one or more oxidases, and biochemical characterization of individual oxidases. Terminal oxidases that share the essential properties of substrate (cytochrome c or quinol) oxidation, dioxygen reduction and, in some cases, proton translocation, differ in subunit architecture and complement of redox centres. Perhaps more significantly, they differ in their affinities for oxidant and reductant, mode of regulation, and inhibitor sensitivity; these differences to some extent rationalize the presence of multiple oxidases. However, intriguing requirements for particular functions in certain physiological functions remain unexplained. For example, a large body of evidence demonstrates that cytochrome bd is essential for growth and survival under certain conditions. In this review, the physiological basis of the many phenotypes of Cyd-mutants is explored, particularly the requirement for this oxidase in diazotrophy, growth at low protonmotive force, survival in the stationary phase, and resistance to oxidative stress and Fe(III) chelators.