Redox and Chemical Activities of the Hemes in the Sulfur Oxidation Pathway Enzyme SoxAX

SoxAX enzymes couple disulfide bond formation to the reduction of cytochrome c in the first step of the phylogenetically widespread Sox microbial sulfur oxidation pathway. Rhodovulum sulfidophilum SoxAX contains three hemes. An electrochemical cell compatible with magnetic circular dichroism at near infrared wavelengths has been developed to resolve redox and chemical properties of the SoxAX hemes. In combination with potentiometric titrations monitored by electronic absorbance and EPR, this method defines midpoint potentials (E_m) at pH 7.0 of approximately +210, −340, and −400 mV for the His/Met, His/Cys⁻, and active site His/CysS⁻-ligated heme, respectively. Exposing SoxAX to S₂O₄²⁻, a substrate analog with E_m ∼−450 mV, but not Eu(II) complexed with diethylene triamine pentaacetic acid (E_m ∼−1140 mV), allows cyanide to displace the cysteine persulfide (CysS⁻) ligand to the active site heme. This provides the first evidence for the dissociation of CysS⁻ that has been proposed as a key event in SoxAX catalysis.     

Effect of cyanide in dark and light on the membrane potential and the ATP level of young and mature green tissues of higher plants

The effect of CN⁻ and N₂ on the electrical membrane potential (E_m) was compared with that of CN⁻ on the ATP levels in cotyledons of Gossypium hirsutum and in Lemna gibba L. In mature cotton tissue, CN⁻ depolarized E_m to the energy-independent diffusion potential (E_D) in the dark. In the light E_m recovered transiently. The same was observed in leaves of Nicotiana, Avena, Impatiens, Kalanchoë, and in Lemna. In contrast, in young cotton cotyledons and tobacco leaves and, to a large extent, in +sucrose-grown Lemna, E_m was depolarized to E_D also in the light in a similar way as in the dark.

In Lemna grown without sucrose, the energy-dependent component of E_m was only partially depolarized by CN⁻ in dark or light. Cyanide plus salicylhydroxamic acid completely reduced E_m to E_D, abolished respiration and photosynthesis, and severely diminished the ATP level. This suggests the operation of a CN⁻-insensitive respiration in uninjured Lemna. The initial CN⁻-induced decay of the ATP level in cotton and Lemna was more rapid than the decay of E_m. CN⁻-induced oscillations of the ATP level were followed by similar but slower oscillations of E_m. This supports the view of a general dependence of E_m on ATP. Discrepancies between inhibitor-induced changes of E_m and ATP levels are suggested to result from additional regulation of E_m by the cytoplasmatic pH value.

A comparison of E_D in young and mature cotton cotyledons in the dark and in the light suggests that in growing young cotyledons the different effect of CN⁻ in the light is due to a less effective photosynthesis together with high mitochondrial respiration. In Lemna and in mature cotton tissue, E_m in the light is maintained by noncyclic photophosphorylation and photosystem II, which is only partly inhibited by CN⁻, thus resulting in an incomplete depolarization and recovery of E_m. Complete inhibition of photosynthetic O₂ evolution and membrane depolarization by CN⁻ plus salicylhydroxamic acid are suggested to result from photooxidation.

     

Mitochondrial Networks in Cardiac Myocytes Reveal Dynamic Coupling Behavior

Oscillatory behavior of mitochondrial inner membrane potential (ΔΨ_m) is commonly observed in cells subjected to oxidative or metabolic stress. In cardiac myocytes, the activation of inner membrane pores by reactive oxygen species (ROS) is a major factor mediating intermitochondrial coupling, and ROS-induced ROS release has been shown to underlie propagated waves of ΔΨ_m depolarization as well as synchronized limit cycle oscillations of ΔΨ_m in the network. The functional impact of ΔΨ_m instability on cardiac electrophysiology, Ca²⁺ handling, and even cell survival, is strongly affected by the extent of such intermitochondrial coupling. Here, we employ a recently developed wavelet-based analytical approach to examine how different substrates affect mitochondrial coupling in cardiac cells, and we also determine the oscillatory coupling properties of mitochondria in ventricular cells in intact perfused hearts. The results show that the frequency of ΔΨ_m oscillations varies inversely with the size of the oscillating mitochondrial cluster, and depends on the strength of local intermitochondrial coupling. Time-varying coupling constants could be quantitatively determined by applying a stochastic phase model based on extension of the well-known Kuramoto model for networks of coupled oscillators. Cluster size-frequency relationships varied with different substrates, as did mitochondrial coupling constants, which were significantly larger for glucose (7.78 × 10⁻² ± 0.98 × 10⁻² s⁻¹) and pyruvate (7.49 × 10⁻² ± 1.65 × 10⁻² s⁻¹) than lactate (4.83 × 10⁻² ± 1.25 × 10⁻² s⁻¹) or β-hydroxybutyrate (4.11 × 10⁻² ± 0.62 × 10⁻² s⁻¹). The findings indicate that mitochondrial spatiotemporal coupling and oscillatory behavior is influenced by substrate selection, perhaps through differing effects on ROS/redox balance. In particular, glucose-perfusion generates strong intermitochondrial coupling and temporal oscillatory stability. Pathological changes in specific catabolic pathways, which are known to occur during the progression of cardiovascular disease, could therefore contribute to altered sensitivity of the mitochondrial network to oxidative stress and emergent ΔΨ_m instability, ultimately scaling to produce organ level dysfunction.     

Diversity of Cytochrome bc Complexes: Example of the Rieske Protein in Green Sulfur Bacteria

The Rieske 2Fe2S cluster of Chlorobium limicola forma thiosulfatophilum strain tassajara was studied by electron paramagnetic resonance spectroscopy. Two distinct orientations of its g tensor were observed in oriented samples corresponding to differing conformations of the protein. Only one of the two conformations persisted after treatment with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. A redox midpoint potential (E_m) of +160 mV in the pH range of 6 to 7.7 and a decreasing E_m (−60 to −80 mV/pH unit) above pH 7.7 were found. The implications of the existence of differing conformational states of the Rieske protein, as well as of the shape of its E_m-versus-pH curve, in green sulfur bacteria are discussed.     

Electrogenic transport of protons driven by the plasma membrane ATPase in membrane vesicles from radish : biochemical characterization

Mg:ATP-dependent H⁺ pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Δ ψ). ΔpH was measured as the decrease of absorbance of Acridine orange and Δ ψ as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Δ pH and Δ ψ generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Δ pH generation is not inhibited by NO₃⁻. These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H⁺-ATPases. Both intravesicular acidification and Δ ψ generation are influenced by anions: Δ pH increases and Δ ψ decreases following the sequence SO₄²⁻, Cl⁻, Br⁻, NO₃⁻. ATP-dependent H⁺ pumping strictly requires Mg²⁺. It is very specific for ATP (apparent K_m 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Δ pH generation is inhibited by CuSO₄ and diethylstilbestrol as well as vanadate. Δ pH generation is specificially stimulated by K⁺ (+ 80%) and to a lesser extent by Na⁺ and choline (+28% and +14%, respectively). The characteristics of H⁺ pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials.     

Characterization of Dye-decolorizing Peroxidase (DyP) from Thermomonospora curvata Reveals Unique Catalytic Properties of A-type DyPs

Dye-decolorizing peroxidases (DyPs) comprise a new family of heme peroxidases, which has received much attention due to their potential applications in lignin degradation. A new DyP from Thermomonospora curvata (TcDyP) was identified and characterized. Unlike other A-type enzymes, TcDyP is highly active toward a wide range of substrates including model lignin compounds, in which the catalytic efficiency with ABTS (k_cat^app/K_m^app = (1.7 × 10⁷) m⁻¹ s⁻¹) is close to that of fungal DyPs. Stopped-flow spectroscopy was employed to elucidate the transient intermediates as well as the catalytic cycle involving wild-type (wt) and mutant TcDyPs. Although residues Asp²²⁰ and Arg³²⁷ are found necessary for compound I formation, His³¹² is proposed to play roles in compound II reduction. Transient kinetics of hydroquinone (HQ) oxidation by wt-TcDyP showed that conversion of the compound II to resting state is a rate-limiting step, which will explain the contradictory observation made with the aspartate mutants of A-type DyPs. Moreover, replacement of His³¹² and Arg³²⁷ has significant effects on the oligomerization and redox potential (E°′) of the enzyme. Both mutants were found to promote the formation of dimeric state and to shift E°′ to a more negative potential. Not only do these results reveal the unique catalytic property of the A-type DyPs, but they will also facilitate the development of these enzymes as lignin degraders.     

Protein Crystallography Reveals a Role for the FS0 Cluster of Escherichia coli Nitrate Reductase A (NarGHI) in Enzyme Maturation

We have used site-directed mutagenesis, EPR spectroscopy, redox potentiometry, and protein crystallography to monitor assembly of the FS0 [4Fe-4S] cluster and molybdo-bis(pyranopterin guanine dinucleotide) cofactor (Mo-bisPGD) of the Escherichia coli nitrate reductase A (NarGHI) catalytic subunit (NarG). Cys and Ser mutants of NarG-His⁴⁹ both lack catalytic activity, with only the former assembling FS0 and Mo-bisPGD. Importantly, both prosthetic groups are absent in the NarG-H49S mutant. EPR spectroscopy of the Cys mutant reveals that the E_m value of the FS0 cluster is decreased by at least 500 mV, preventing its participation in electron transfer to the Mo-bisPGD cofactor. To demonstrate that decreasing the FS0 cluster E_m results in decreased enzyme activity, we mutated a critical Arg residue (NarG-Arg⁹⁴) in the vicinity of FS0 to a Ser residue. In this case, the E_m of FS0 is decreased by 115 mV, with a concomitant decrease in enzyme turnover to ∼30% of the wild type. Analysis of the structure of the NarG-H49S mutant reveals two important aspects of NarGHI maturation: (i) apomolybdo-NarGHI is able to bind GDP moieties at their respective P and Q sites in the absence of the Mo-bisPGD cofactor, and (ii) a critical segment of residues in NarG, ⁴⁹HGVNCTG⁵⁵, must be correctly positioned to ensure holoenzyme maturation.     

The NADP-Dependent Methylene Tetrahydromethanopterin Dehydrogenase in Methylobacterium extorquens AM1

An NADP-dependent methylene tetrahydromethanopterin (H₄MPT) dehydrogenase has recently been proposed to be involved in formaldehyde oxidation to CO₂ in Methylobacterium extorquens AM1. We report here on the purification of this novel enzyme to apparent homogeneity. Via the N-terminal amino acid sequence, it was identified to be the mtdA gene product. The purified enzyme catalyzed the dehydrogenation of methylene H₄MPT with NADP⁺ rather than with NAD⁺, with a specific activity of approximately 400 U/mg of protein. It also catalyzed the dehydrogenation of methylene tetrahydrofolate (methylene H₄F) with NADP⁺. With methylene H₄F as the substrate, however, the specific activity (26 U/mg) and the catalytic efficiency (V_max/K_m) were approximately 20-fold lower than with methylene H₄MPT. Whereas the dehydrogenation of methylene H₄MPT (E₀ = −390 mV) with NADP⁺ (E₀ = −320 mV) proceeded essentially irreversibly, the dehydrogenation of methylene H₄F (E₀ = −300 mV) was fully reversible. Comparison of the primary structure of the NADP-dependent dehydrogenase from M. extorquens AM1 with those of methylene H₄F dehydrogenases from other bacteria and eucarya and with those of methylene H₄MPT dehydrogenases from methanogenic archaea revealed only marginally significant similarity (<15%).     

Thyroid Hormone Signaling In Vivo Requires a Balance between Coactivators and Corepressors

Resistance to thyroid hormone (RTH), a human syndrome, is characterized by high thyroid hormone (TH) and thyroid-stimulating hormone (TSH) levels. Mice with mutations in the thyroid hormone receptor beta (TRβ) gene that cannot bind steroid receptor coactivator 1 (SRC-1) and Src-1^−/− mice both have phenotypes similar to that of RTH. Conversely, mice expressing a mutant nuclear corepressor 1 (Ncor1) allele that cannot interact with TRβ, termed NCoRΔID, have low TH levels and normal TSH. We hypothesized that Src-1^−/− mice have RTH due to unopposed corepressor action. To test this, we crossed NCoRΔID and Src-1^−/− mice to create mice deficient for coregulator action in all cell types. Remarkably, NCoR^ΔID/ΔID Src-1^−/− mice have normal TH and TSH levels and are triiodothryonine (T₃) sensitive at the level of the pituitary. Although absence of SRC-1 prevented T₃ activation of key hepatic gene targets, NCoR^ΔID/ΔID Src-1^−/− mice reacquired hepatic T₃ sensitivity. Using in vivo chromatin immunoprecipitation assays (ChIP) for the related coactivator SRC-2, we found enhanced SRC-2 recruitment to TR-binding regions of genes in NCoR^ΔID/ΔID Src-1^−/− mice, suggesting that SRC-2 is responsible for T₃ sensitivity in the absence of NCoR1 and SRC-1. Thus, T₃ targets require a critical balance between NCoR1 and SRC-1. Furthermore, replacement of NCoR1 with NCoRΔID corrects RTH in Src-1^−/− mice through increased SRC-2 recruitment to T₃ target genes.     

tRNAHis 5-methylcytidine levels increase in response to several growth arrest conditions in Saccharomyces cerevisiae

tRNAs are highly modified, each with a unique set of modifications. Several reports suggest that tRNAs are hypomodified or, in some cases, hypermodified under different growth conditions and in certain cancers. We previously demonstrated that yeast strains depleted of tRNA^His guanylyltransferase accumulate uncharged tRNA^His lacking the G₋₁ residue and subsequently accumulate additional 5-methylcytidine (m⁵C) at residues C₄₈ and C₅₀ of tRNA^His, due to the activity of the m⁵C-methyltransferase Trm4. We show here that the increase in tRNA^His m⁵C levels does not require loss of Thg1, loss of G₋₁ of tRNA^His, or cell death but is associated with growth arrest following different stress conditions. We find substantially increased tRNA^His m⁵C levels after temperature-sensitive strains are grown at nonpermissive temperature, and after wild-type strains are grown to stationary phase, starved for required amino acids, or treated with rapamycin. We observe more modest accumulations of m⁵C in tRNA^His after starvation for glucose and after starvation for uracil. In virtually all cases examined, the additional m⁵C on tRNA^His occurs while cells are fully viable, and the increase is neither due to the GCN4 pathway, nor to increased Trm4 levels. Moreover, the increased m⁵C appears specific to tRNA^His, as tRNA^Val(AAC) and tRNA^Gly(GCC) have much reduced additional m⁵C during these growth arrest conditions, although they also have C₄₈ and C₅₀ and are capable of having increased m⁵C levels. Thus, tRNA^His m⁵C levels are unusually responsive to yeast growth conditions, although the significance of this additional m⁵C remains unclear.     

Controls of Evapotranspiration and CO2 Fluxes from Scots Pine by Surface Conductance and Abiotic Factors

Evapotranspiration (E) and CO₂ flux (F_c) in the growing season of an unusual dry year were measured continuously over a Scots pine forest in eastern Finland, by eddy covariance techniques. The aims were to gain an understanding of their biological and environmental control processes. As a result, there were obvious diurnal and seasonal changes in E, F_c, surface conductance (g_c), and decoupling coefficient (Ω), showing similar trends to those in radiation (PAR) and vapour pressure deficit (δ). The maximum mean daily values (24-h average) for E, F_c, g_c, and Ω were 1.78 mmol m⁻² s⁻¹, −11.18 µmol m⁻² s⁻¹, 6.27 mm s⁻¹, and 0.31, respectively, with seasonal averages of 0.71 mmol m⁻² s⁻¹, −4.61 µmol m⁻² s⁻¹, 3.3 mm s⁻¹, and 0.16. E and F_c were controlled by combined biological and environmental variables. There was curvilinear dependence of E on g_c and F_c on g_c. Among the environmental variables, PAR was the most important factor having a positive linear relationship to E and curvilinear relationship to F_c, while vapour pressure deficit was the most important environmental factor affecting g_c. Water use efficiency was slightly higher in the dry season, with mean monthly values ranging from 6.67 to 7.48 μmol CO₂ (mmol H₂O)⁻¹ and a seasonal average of 7.06 μmol CO₂ (μmol H₂O)⁻¹. Low Ω and its close positive relationship with g_c indicate that evapotranspiration was sensitive to surface conductance. Mid summer drought reduced surface conductance and decoupling coefficient, suggesting a more biotic control of evapotranspiration and a physiological acclimation to dry air. Surface conductance remained low and constant under dry condition, supporting that a constant value of surface constant can be used for modelling transpiration under drought condition.     

Effects of temperature on orthophosphate absorption by excised corn roots

The uptake of orthophosphate (³²P) by excised corn roots, Zea mays L. was studied using roots grown on 0.2 mm CaSO₄. Nine concentrations of KH₂PO₄ from 1 to 256 μm were used at temperatures of 20°, 30°, and 40°. Enzyme kinetic analysis was applied to the data obtained. Two apparent mechanisms (sites) of phosphate uptake were observed, 1 dominating at high P concentrations and 1 at low P concentrations. A Km of 1.36 × 10⁻⁴ and a Vmax of 177 × 10⁻⁹ moles per gram of roots per hour at 30° was calculated for the mechanism dominating at high P concentrations. Similar calculations gave a Km of 6.09 × 10⁻⁶ and a Vmax of 162 × 10⁻⁹ moles per gram of roots per hour at 30° for the mechanism dominating at low P concentrations. The Q₁₀ for both mechanisms was approximately 2. Calculation of thermodynamic values from the data gave ΔF of − 5200 cal, ΔH of − 950 to − 1400 cal, and a enthalpy of activation (A) of 10,300 to 13,800 cal per mole for the mechanism dominating at high P concentrations. Similar calculations from the data for the mechanism dominating at low P concentrations gave a ΔF of − 7300 cal, ΔH of − 10,700 to − 8200 cal, and a A of 9300 to 18,900 cal per mole. If the dual mechanism interpretation of this kind of data adequately describes this system, then both mechanisms of P absorption by corn roots involve chemical reactions.     

Influence of Acidic pH on Hydrogen and Acetate Production by an Electrosynthetic Microbiome

Production of hydrogen and organic compounds by an electrosynthetic microbiome using electrodes and carbon dioxide as sole electron donor and carbon source, respectively, was examined after exposure to acidic pH (∼5). Hydrogen production by biocathodes poised at −600 mV vs. SHE increased>100-fold and acetate production ceased at acidic pH, but ∼5–15 mM (catholyte volume)/day acetate and>1,000 mM/day hydrogen were attained at pH ∼6.5 following repeated exposure to acidic pH. Cyclic voltammetry revealed a 250 mV decrease in hydrogen overpotential and a maximum current density of 12.2 mA/cm² at −765 mV (0.065 mA/cm² sterile control at −800 mV) by the Acetobacterium-dominated community. Supplying −800 mV to the microbiome after repeated exposure to acidic pH resulted in up to 2.6 kg/m³/day hydrogen (≈2.6 gallons gasoline equivalent), 0.7 kg/m³/day formate, and 3.1 kg/m³/day acetate ( = 4.7 kg CO₂ captured).     

How Periplasmic Thioredoxin TlpA Reduces Bacterial Copper Chaperone ScoI and Cytochrome Oxidase Subunit II (CoxB) Prior to Metallation

Two critical cysteine residues in the copper-A site (Cu_A) on subunit II (CoxB) of bacterial cytochrome c oxidase lie on the periplasmic side of the cytoplasmic membrane. As the periplasm is an oxidizing environment as compared with the reducing cytoplasm, the prediction was that a disulfide bond formed between these cysteines must be eliminated by reduction prior to copper insertion. We show here that a periplasmic thioredoxin (TlpA) acts as a specific reductant not only for the Cu²⁺ transfer chaperone ScoI but also for CoxB. The dual role of TlpA was documented best with high-resolution crystal structures of the kinetically trapped TlpA-ScoI and TlpA-CoxB mixed disulfide intermediates. They uncovered surprisingly disparate contact sites on TlpA for each of the two protein substrates. The equilibrium of CoxB reduction by TlpA revealed a thermodynamically favorable reaction, with a less negative redox potential of CoxB (E′₀ = −231 mV) as compared with that of TlpA (E′₀ = −256 mV). The reduction of CoxB by TlpA via disulfide exchange proved to be very fast, with a rate constant of 8.4 × 10⁴ m⁻¹ s⁻¹ that is similar to that found previously for ScoI reduction. Hence, TlpA is a physiologically relevant reductase for both ScoI and CoxB. Although the requirement of ScoI for assembly of the Cu_A-CoxB complex may be bypassed in vivo by high environmental Cu²⁺ concentrations, TlpA is essential in this process because only reduced CoxB can bind copper ions.     

In Vivo 23Na Nuclear Magnetic Resonance Study of Maintenance of a Sodium Gradient in the Ruminal Bacterium Fibrobacter succinogenes S85

Sodium gradients (ΔpNa) were measured in resting cells of Fibrobacter succinogenes by in vivo ²³Na nuclear magnetic resonance using Tm(DOTP)⁵⁻ [thulium(III) 1,4,7,10-tetraazacyclododecane-N′,N′′,N′′′-tetramethylenephosphonate] as the shift reagent. This bacterium was able to maintain a ΔpNa of −55 to −40 mV for extracellular sodium concentrations ranging from 30 to 200 mM. Depletion of Na⁺ ions during the washing steps led to irreversible damage (modification of glucose metabolism and inability to maintain a sodium gradient).     

Potential-dependent anion transport in tonoplast vesicles from oat roots

Potential-dependent anion movement into tonoplast vesicles from oat roots (Avena sativa L. var Lang) was monitored as dissipation of membrane potentials (Δψ) using the fluorescence probe Oxonol V. The potentials (positive inside) were generated with the H⁺-pumping pyrophosphatase, which is K⁺ stimulated and anion insensitive. The relative rate of ΔΨ dissipation by anions was used to estimate the relative permeabilities of the anions. In decreasing order they were: SCN⁻ (100) > NO₃⁻ (72) = Cl⁻ (70) > Br⁻ (62) > SO₄²⁻ (5) = H₂PO₄⁻ (5) > malate (3) = acetate (3) > iminodiacetate (2). Kinetic studies showed that the rate of Δψ dissipation by Cl⁻ and NO₃⁻, but not by SCN⁻, was saturable. The K_m values for Cl⁻ and NO₃⁻ uptake were about 2.3 and 5 millimolar, respectively, suggesting these anions move into the vacuole through proteinaceous porters. In contrast to a H⁺-coupled Cl⁻ transporter on the same vesicles, the potential-dependent Cl⁻ transport was insensitive to 4,4′-diisothiocyano-2,2′-stilbene disulfonate. These results suggest the existence of at least two different mechanisms for Cl⁻ transport in these vesicles. The potentials generated by the H⁺-translocating ATPase and H⁺-pyrophosphatase were nonadditive, giving support to the model that both pumps are on tonoplast vesicles. No evidence for a putative Cl⁻ conductance on the anion-sensitive H⁺-ATPase was found.     

Electrogenic ATPase Activity on the Peribacteroid Membrane of Soybean (Glycine max L.) Root Nodules

Electrogenic ATPase activity on the peribacteroid membrane from soybean (Glycine max L. cv Bragg) root nodules is demonstrated. Membrane energization was monitored using suspensions of intact peribacteroid membrane-enclosed bacteroids (peribacteroid units; PBUs) and the fluorescent probe for membrane potential (ΔΨ), bis-(3-phenyl-5-oxoisoxazol-4yl) pentamethine oxonol. Generation of a positive ΔΨ across the peribacteroid membrane was dependent upon ATP, inhibited by N,N′-dicyclohexyl-carbodiimide and vanadate, but insensitive to N-ethylmaleimide, azide, cyanide, oligomycin, and ouabain. The results suggest the presence of a single, plasma membrane-like, electrogenic ATPase on the peribacteroid membrane. The protonophore, carbonyl-cyanide m-chlorophenyl hydrazone, completely dissipated the established membrane potential. The extent of reduction in the steady state membrane potential upon addition of ions was used to estimate the relative permeability of the peribacteroid membrane to anions. By this criterion, the relative rates of anion transport across the peribacteroid membrane were: NO₃⁻ > NO₂⁻ > Cl⁻ > acetate⁻ > malate⁻. The observation that 10 millimolar NO₃⁻ completely dissipated the membrane potential was of particular interest in view of the fact that NO₃⁻ inhibits symbiotic nitrogen fixation. The possible function of the ATPase in symbiotic nitrogen fixation is discussed.     

Integrating Terminal Truncation and Oligopeptide Fusion for a Novel Protein Engineering Strategy To Improve Specific Activity and Catalytic Efficiency: Alkaline α-Amylase as a Case Study

In this work, we integrated terminal truncation and N-terminal oligopeptide fusion as a novel protein engineering strategy to improve specific activity and catalytic efficiency of alkaline α-amylase (AmyK) from Alkalimonas amylolytica. First, the C terminus or N terminus of AmyK was partially truncated, yielding 12 truncated mutants, and then an oligopeptide (AEAEAKAKAEAEAKAK) was fused at the N terminus of the truncated AmyK, yielding another 12 truncation-fusion mutants. The specific activities of the truncation-fusion mutants AmyKΔC500-587::OP and AmyKΔC492-587::OP were 25.5- and 18.5-fold that of AmyK, respectively. The k_cat/K_m was increased from 1.0 × 10⁵ liters · mol⁻¹ · s⁻¹ for AmyK to 30.6 × and 23.2 × 10⁵ liters · mol⁻¹ · s⁻¹ for AmyKΔC500-587::OP and AmyKΔC492-587::OP, respectively. Comparative analysis of structure models indicated that the higher flexibility around the active site may be the main reason for the improved catalytic efficiency. The proposed terminal truncation and oligopeptide fusion strategy may be effective to engineer other enzymes to improve specific activity and catalytic efficiency.